Glucagon/glp-1 receptor co-agonists

ABSTRACT

Modified glucagon peptides are disclosed having enhanced potency at the glucagon receptor relative to native glucagon. Further modification of the glucagon peptides by forming lactam bridges or the substitution of the terminal carboxylic acid with an amide group produces peptides exhibiting glucagon/GLP-1 receptor co-agonist activity. The solubility and stability of these high potency glucagon analogs can be further improved by modification of the polypeptides by pegylation, substitution of carboxy terminal amino acids, or the addition of a carboxy terminal peptide selected from the group consisting of SEQ ID NO: 26 (GPSSGAPPPS), SEQ ID NO: 27 (KRNRNNIA) and SEQ ID NO: 28 (KRNR).

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 60/890,087 filed on Feb. 15, 2007 and U.S. Provisional PatentApplication No. 60/938,565 filed May 17, 2007. The subject matterdisclosed in these provisional applications is hereby expresslyincorporated by reference into the present application.

BACKGROUND

Pre-proglucagon is a 158 amino acid precursor polypeptide that isprocessed in different tissues to form a number of differentproglucagon-derived peptides, including glucagon, glucagon-likepeptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2) and oxyntomodulin(OXM), that are involved in a wide variety of physiological functions,including glucose homeostasis, insulin secretion, gastric emptying, andintestinal growth, as well as the regulation of food intake. Glucagon isa 29-amino acid peptide that corresponds to amino acids 33 through 61 ofpre-proglucagon, while GLP-1 is produced as a 37-amino acid peptide thatcorresponds to amino acids 72 through 108 of pre-proglucagon.GLP-1(7-36) amide or GLP-1(7-37) acid are biologically potent fool's ofGLP-1, that demonstrate essentially equivalent activity at the GLP-1receptor.

Hypoglycemia occurs when blood glucose levels drops too low to provideenough energy for the body's activities. In adults or children olderthan 10 years, hypoglycemia is uncommon except as a side effect ofdiabetes treatment, but it can result from other medications ordiseases, hormone or enzyme deficiencies, or tumors. When blood glucosebegins to fall, glucagon, a hormone produced by the pancreas, signalsthe liver to break down glycogen and release glucose, causing bloodglucose levels to rise toward a normal level. Thus, glucagon's generalrole in glucose regulation is to'counteract the action of insulin andmaintain blood glucose levels. However for diabetics, this glucagonresponse to hypoglycemia may be impaired, making it harder for glucoselevels to return to the normal range.

Hypoglycemia is a life threatening event that requires immediate medicalattention. The administration of glucagon is an established Medicationfor treating acute hypoglycemia and it can restore normal levels ofglucose within, minutes of administration. When glucagon is used in theacute medical treatment of hypoglycemia, a crystalline form of glucagonis solubilized with a dilute acid buffer and the solution is injectedintramuscularly. While this treatment is effective, the methodology iscumbersome and dangerous for someone that is semi-conscious.Accordingly, there is a need for a glucagon analog that maintains orexceeds the biological performance of the parent molecule but issufficiently soluble and stable, under relevant physiologicalconditions, that it can be pre-formulated as a solution, ready forinjection.

Additionally, diabetics are encouraged to maintain near normal bloodglucose levels to delay or prevent microvascular complications.Achievement of this goal usually requires intensive insulin therapy. Instriving to achieve this goal, physicians have encountered a substantialincrease in the frequency and severity of hypoglycemia in their diabeticpatients. Accordingly, improved pharmaceuticals and methodologies areneeded for treating diabetes that are less likely to induce hypoglycemiathan current insulin therapies.

GLP-1 has different biological activities compared to glucagon. Itsactions include stimulation of insulin synthesis and secretion,inhibition of glucagon secretion, and inhibition of food intake. GLP-1has been shown to reduce hyperglycemia (elevated glucose levels) indiabetics. Exendin-4, a peptide from lizard venom that shares about 50%amino acid identity with GLP-1, activates the GLP-1 receptor andlikewise has been shown to reduce hyperglycemia in diabetics.

There is also evidence that GLP-1 and exendin-4 may reduce food intakeand promote weight loss, an effect that would be beneficial not only fordiabetics but also for patients suffering from obesity. Patients withobesity have a higher risk of diabetes, hypertension, hyperlipidemia,cardiovascular disease, and musculoskeletal diseases.

Accordingly, there remains a need for alternative and preferablyimproved methods for treating diabetes and obesity.

SUMMARY

As described herein, high potency glucagon agonists analogs are providedthat also exhibit increased activity at the glucagon receptor, and infurther embodiments exhibit enhanced biophysical stability and/oraqueous solubility. In addition, in accordance with another aspect ofthe invention, glucagon agonist analogs are provided that have lostnative glucagon's selectivity for the glucagon receptor verses the GLP-1receptor, and thus represent co-agonists of those two receptors.Selected amino acid modifications within the glucagon analogs cancontrol the relative activity of the analog at the GLP-1 receptor versesthe glucagon receptor. Thus, yet another aspect of the inventionprovides glucagon co-agonist analogs that have higher activity at theglucagon receptor versus the GLP-1 receptor, glucagon co-agonist analogsthat have approximately equivalent activity at both receptors, andglucagon co-agonist analogs that have higher activity at the GLP-1receptor versus the glucagon receptor. The latter category of co-agonistcan be engineered to exhibit little or no activity at the glucagonreceptor, and yet retain ability to activate the GLP-1 receptor with thesame or better potency than native GLP-1. Any of these analogs may alsoinclude modifications that confer enhanced biophysical stability and/oraqueous solubility.

Glucagon analogs that demonstrate co-agonism at the glucagon and GLP-1receptors are advantageous for several applications. First of all theuse of glucagon to treat hypoglycemia may overcompensate for low bloodglucose levels and result in excess blood glucose levels. If aglucagon/GLP-1 receptor co-agonist is administered, the additional GLP-1stimulation may buffer the glucagon agonist effect to prevent excessiveglucose blood levels resulting from treatment of hypoglycemia.

In addition as described herein, glucagon co-agonist analogs of theinvention may be used to control hyperglycemia, or to induce weight lossor prevent weight gain, when administered alone or in combination withother anti-diabetic or anti-obesity treatments. Another compound thatinduces weight loss is oxyntomodulin, a naturally occurring digestivehormone found in the small intestine (see Diabetes 2005; 54:2390-2395).Oxyntomodulin is a 37 amino acid peptide that contains the 29 amino acidsequence of glucagon (i.e. SEQ ID NO: 1) followed by an 8 amino acidcarboxy terminal extension of SEQ ID NO: 27 (KRNRNNIA). While thepresent invention contemplates that glucagon analogs described hereinmay optionally be joined to this 8 amino acid carboxy terminal extension(SEQ ID NO: 27), the invention in some embodiments also specificallycontemplates analogs and uses of analogs lacking the 8 contiguouscarboxy amino acids of SEQ ID NO: 27.

The compounds can be customized by amino acid modifications to regulatethe GLP-1 activity of the peptide, and thus the glucagon analogs of thepresent can be tailored to treat a particular condition or disease. Moreparticularly, glucagon analogs are provided herein wherein each analogdisplays a characteristic relative level of activity at the respectiveglucagon and GLP-1 receptors. For example, modifications can be made toeach peptide to produce a glucagon peptide having anywhere from at leastabout 10% (including at least about 20%, 30%, 40%, 50%, 60%, 75%, 100%,125%, 150%, 175%) to about 200% or higher activity at the GLP-1 receptorrelative to native GLP-1 and anywhere from at least about 10% (includingabout 20%, 30%, 40%, 50%, 60%, 75%, 100%, 125%, 150%, 175%, 200%, 250%,300%, 350%, 400%, 450%) to about 500% or higher activity at the glucagonreceptor relative to native glucagon. The amino acid sequence of nativeglucagon is SEQ ID NO: 1, the amino acid sequence of GLP-1(7-36)amide isSEQ ID NO: 52, and the amino acid sequence of GLP-1(7-37) acid is SEQ IDNO: 50. In exemplary embodiments, a glucagon peptide may exhibit atleast 10% of the activity of native glucagon at the glucagon receptorand at least 50% of the activity of native GLP-1 at the GLP-1 receptor,or at least 40% of the activity of native glucagon at the glucagonreceptor and at least 40% of the activity of native GLP-1 at the GLP-1receptor, or at least 60% of the activity of native glucagon at theglucagon receptor and at least 60% of the activity of native GLP-1 atthe GLP-1 receptor.

Selectivity of a glucagon peptide for the glucagon receptor versus theGLP-1 receptor can be described as the relative ratio of glucagon/GLP-1activity (the peptide's activity at the glucagon receptor relative tonative glucagon, divided by the peptide's activity at the GLP-1 receptorrelative to native GLP-1). For example, a glucagon peptide that exhibits60% of the activity of native glucagon at the glucagon receptor and 60%of the activity of native GLP-1 at the GLP-1 receptor has a 1:1 ratio ofglucagon/GLP-1 activity. Exemplary ratios of glucagon/GLP-1 activityinclude about 1:1, 1.5:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or10:1, or about 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, or 1:1.5.As an example, a glucagon/GLP-1 activity ratio of 10:1 indicates a10-fold selectivity for the glucagon receptor versus the GLP-1 receptor.Similarly, a GLP-1/glucagon activity ratio of 10:1 indicates a 10-foldselectivity for the GLP-1 receptor versus the glucagon receptor.

In accordance with one embodiment, analogs of glucagon are provided thathave enhanced potency and optionally improved solubility and stability.In one embodiment, enhanced glucagon potency is provided by an aminoacid modification at position 16 of native glucagon (SEQ ID NO: 1). Byway of nonlimiting example, such enhanced potency can be provided bysubstituting the naturally occurring serine at position 16 with glutamicacid or with another negatively charged amino acid having a side chainwith a length of 4 atoms, or alternatively with any one of glutamine,homoglutamic acid, or homocysteic acid, or a charged amino acid having aside chain containing at least one heteroatom, (e.g. N, O, S, P) andwith a side chain length of about 4 (or 3-5) atoms. In one embodimentthe enhanced potency glucagon agonist comprises a peptide of SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7or a glucagon agonist analog of SEQ ID NO: 5. In accordance with oneembodiment a glucagon analog protein having enhanced potency at theglucagon receptor relative to wild type glucagon is provided wherein thepeptide comprises the sequence of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO:9 or SEQ ID NO: 10, wherein the glucagon peptide retains its selectivityfor the glucagon receptor relative to the GLP-1 receptors.

Glucagon receptor activity can be reduced by an amino acid modificationat position 3, e.g. substitution of the naturally occurring glutamine atposition 3 with any amino acid. Substitution at this position with anacidic, basic, or a hydrophobic amino acid (glutamic acid, ornithine,norleucine) has been shown to substantially reduce or destroy glucagonreceptor activity. In some embodiments the analogs have about 10% orless of the activity of native glucagon at the glucagon receptor, e.g.about 1-10%, or about 0.1-10%, or greater than about 0.1% but less thanabout 10%, while exhibiting at least 20% of the activity of GLP-1 at theGLP-1 receptor. For example, exemplary analogs described herein haveabout 0.5%, about 1% or about 7% of the activity of native glucagon,while exhibiting at least 20% of the activity of GLP-1 at the GLP-1receptor.

In another embodiment analogs of glucagon are provided that haveenhanced or retained potency at the glucagon receptor relative to thenative glucagon peptide, but also have greatly enhanced activity at theGLP-1 receptor. Glucagon normally has about 1% of the activity ofnative-GLP-1 at the GLP-1 receptor, while GLP-1 normally has less thanabout 0.01% of the activity of native glucagon at the glucagon receptor.Enhanced activity at the GLP-1 receptor is provided by replacing thecarboxylic acid of the C-terminal amino acid with a charge-neutralgroup, such as an amide or ester. In one embodiment, these glucagonanalogs comprise a sequence of SEQ ID NO: 20 wherein the carboxyterminal amino acid has an amide group in place of the carboxylic acidgroup found on the native amino acid. These glucagon analogs have strongactivity at both the glucagon and GLP-1 receptors and thus act asco-agonists at both receptors. In accordance with one embodiment aglucagon and GLP-1 receptor co-agonist is provided wherein the peptidecomprises the sequence of SEQ ID NO: 20, wherein the amino acid atposition 28 is Asn or Lys and the amino acid at position 29 isThr-amide.

Enhanced activity at the GLP-1 receptor is also provided by stabilizingthe alpha-helix structure in the C-terminal portion of glucagon (aroundamino acids 12-29), through formation of an intramolecular bridgebetween the side chains of two amino acids that are separated by threeintervening amino acids. In exemplary embodiments, the bridge or linkeris about 8 (or about 7-9) atoms in length and forms between side chainsof amino acids at positions 12 and 16, or at positions 16 and 20, or atpositions 20 and 24, or at positions 24 and 28. The side chains of theseamino acids can be linked to one another through hydrogen-bonding orionic interactions, such as the formation of salt bridges, or bycovalent bonds. In accordance with one embodiment a glucagon agonist isprovided comprising a glucagon peptide of SEQ ID NO: 20, wherein alactam ring is formed between the side chains of a lysine residue,located at position 12, 20 or 28, and a glutamic acid residue, locatedat position 16 or 24, wherein the two amino acids of the glucagonpeptide whose side chains participate in forming the lactam ring arespaced from one another by three intervening amino acids. In accordancewith one embodiment the lactam bearing glucagon analog comprises anamino acid sequence selected from the group consisting of SEQ ID NO: 11,SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO: 17 and SEQ ID NO: 18. In one embodiment the carboxyterminal amino acid of the lactam bearing peptide comprises an amidegroup or an ester group in place of the terminal carboxylic acid. In oneembodiment a glucagon peptide of SEQ ID NO: 11, SEQ ID NO: 12, SEQ IDNO: 13, and SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17and SEQ ID NO: 18 further comprises an additional amino acid covalentlybound to the carboxy terminus of SEQ ID NO: 11, SEQ ID NO: 12, SEQ IDNO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 orSEQ ID NO: 18. In a further embodiment a glucagon peptide is providedcomprising a sequence selected from the group consisting of SEQ ID NO:66, SEQ ID NO: 67, SEQ ID NO: 68 and SEQ ID NO: 69 further comprises anadditional amino acid covalently bound to the carboxy terminus of SEQ IDNO: 66, SEQ ID NO: 67, SEQ ID NO: 68 and SEQ ID NO: 69. In oneembodiment the amino acid at position 28 is asparagine or lysine and theamino acid at position 29 is threonine.

Enhanced activity at the GLP-1 receptor is also provided by an aminoacid modification at position 20. In one embodiment, the glutamine atposition 20 is replaced with another hydrophilic amino acid having aside chain that is either charged or has an ability to hydrogen-bond,and is at least about 5 (or about 4-6) atoms in length, for example,lysine, citrulline, arginine, or ornithine.

Any of the modifications described above which increase or decreaseglucagon receptor activity and which increase GLP-1 receptor activitycan be applied individually or in combination. Combinations of themodifications that increase GLP-1 receptor activity generally providehigher GLP-1 activity than any of such modifications taken alone. Forexample, the invention provides glucagon analogs that comprisemodifications at position 16, at position 20, and at the C-terminalcarboxylic acid group, optionally with a covalent bond between the aminoacids at positions 16 and 20; glucagon analogs that comprisemodifications at position 16 and at the C-terminal carboxylic acidgroup; glucagon analogs that comprise modifications at positions 16 and20, optionally with a covalent bond between the amino acids at positions16 and 20; and glucagon analogs that comprise modifications at position20 and at the C-terminal carboxylic acid group; optionally with theproviso that the amino acid at position 12 is not Arg; or optionallywith the proviso that the amino acid at position 9 is not Glu.

Other modifications at position 1 or 2, as described herein, canincrease the peptide's resistance to dipeptidyl peptidase IV (DPP IV)cleavage. For example, the amino acid at position 2 may be substitutedwith D-serine, alanine, D-alanine, valine, glycine, N-methyl serine,N-methyl alanine, or amino isobutyric acid. Alternatively, or inaddition, the amino acid at position 1 may be substituted withD-histidine, desaminohistidine, hydroxyl-histidine, acetyl-histidine,homo-histidine, N-methyl histidine, alpha-methyl histidine, imidazoleacetic acid, or alpha, alpha-dimethyl imidiazole acetic acid (DMIA). Itwas observed that modifications at position 2 (e.g. AIB at position 2)and in some cases modifications at position 1 may reduce glucagonactivity, sometimes significantly; surprisingly, this reduction inglucagon activity can be restored by a covalent bond between amino acidsat positions 12 and 16, 16 and 20, or 20 and 24, e.g. a lactam bridgebetween a glutamic acid at position 16 and a lysine at position 20.

In yet further exemplary embodiments, any of the foregoing compounds canbe further modified to improve stability by modifying the amino acid atposition 15 of SEQ ID NO: 1 to reduce degradation of the peptide overtime, especially in acidic or alkaline buffers.

In another embodiment the solubility of the glucagon peptides disclosedherein are enhanced by the covalent linkage of a hydrophilic moiety tothe peptide. In one embodiment the hydrophilic moiety is a polyethyleneglycol (PEG) chain, optionally linked to the peptide at one or more ofpositions 16, 17, 21, 24, 29, or the C-terminus. In some embodiments,the native amino acid at that position is substituted with an amino acidhaving a side chain suitable for crosslinking with hydrophilic moieties,to facilitate linkage of the hydrophilic moiety to the peptide. In otherembodiments, an amino acid modified to comprise a hydrophilic group isadded to the peptide at the C-terminus. In one embodiment the peptideco-agonist comprises a sequence selected from the group consisting ofSEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO:15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19wherein the side chain of an amino acid residue at one of position 16,17, 21 or 24 of said glucagon peptide further comprises a polyethyleneglycol chain, having a molecular weight selected from the range of about500 to about 40,000 Daltons. In one embodiment the polyethylene glycolchain has a molecular weight selected from the range of about 500 toabout 5,000 Daltons. In another embodiment the polyethylene glycol chainhas a molecular weight of about 10,000 to about 20,000 Daltons. In yetother exemplary embodiments the polyethylene glycol chain has amolecular weight of about 20,000 to about 40,000 Daltons.

In another embodiment the solubility of any of the preceding glucagonanalogs can be improved by amino acid substitutions and/or additionsthat introduce a charged amino acid into the C-terminal portion of thepeptide, preferably at a position C-terminal to position 27 of SEQ IDNO: 1. Optionally, one, two or three charged amino acids may beintroduced within the C-terminal portion, preferably C-terminal toposition 27. In accordance with one embodiment the native amino acid(s)at positions 28 and/or 29 are substituted with a charged amino acids,and/or in a further embodiment one to three charged amino acids are alsoadded to the C-terminus of the peptide. In exemplary embodiments, one,two or all of the charged amino acids are negatively charged. Additionalmodifications, e.g. conservative substitutions, may be made to theglucagon peptide that still allow it to retain glucagon activity. In oneembodiment an analog of the peptide of SEQ ID NO: 20 is provided whereinthe analog differs from SEQ ID NO: 20 by 1 to 2 amino acid substitutionsat positions 17-26, and in one embodiment the analog differs from thepeptide of SEQ ID NO: 20 by an amino acid substitution at position 20.

In accordance with one embodiment the glucagon peptides disclosed hereinare modified by the addition of a second peptide to the carboxy teiminusof the glucagon peptide, for example, SEQ ID NO: 26, SEQ ID NO: 27 orSEQ ID NO: 28. In one embodiment a glucagon peptide having a peptidesequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO:12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ IDNO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 66, SEQ ID NO: 67, SEQID NO: 68, and SEQ ID NO: 69 is covalently bound through a peptide bondto a second peptide, wherein the second peptide comprises a sequenceselected from the group consisting of SEQ ID NO: 26, SEQ ID NO: 27 andSEQ ID NO: 28. In a further embodiment, in glucagon peptides whichcomprise the C-terminal extension, the threonine at position 29 of thenative glucagon peptide is replaced with a glycine. A glucagon analoghaving a glycine substitution for threonine at position 29 andcomprising the carboxy terminal extension of SEQ ID NO: 26 is four timesas potent at the GLP-1 receptor as native glucagon modified to comprisethe carboxy terminal extension of SEQ ID NO: 26. Potency at the GLP-1receptor can be further enhanced by an alanine substitution for thenative arginine at position 18.

Thus, as disclosed herein high potency glucagon analogs or glucagonco-agonist analogs are provided that also exhibit improved solubilityand/or stability. An exemplary high potency glucagon analog exhibits atleast about 200% of the activity of native glucagon at the glucagonreceptor, and optionally is soluble at a concentration of at least 1mg/mL at a pH between 6 and 8 (e.g. pH 7), and optionally retains atleast 95% of the original peptide (e.g. 5% or less of the originalpeptide is degraded or cleaved) after 24 hours at 25° C. As anotherexample, an exemplary glucagon co-agonist analog exhibits greater thanabout 40% or greater than about 60% activity at both the glucagon andthe GLP-1 receptors (at a ratio between about 1:3 and 3:1, or betweenabout 1:2 and 2:1), is optionally soluble at a concentration of at least1 mg/mL at a pH between 6 and 8 (e.g. pH 7), and optionally retains atleast 95% of the original peptide after 24 hours at 25° C. Anotherexemplary glucagon co-agonist analog exhibits about 175% or more of theactivity of native glucagon at the glucagon receptor and about 20% orless of the activity of native GLP-1 at the GLP-1 receptor, isoptionally soluble at a concentration of at least 1 mg/mL at a pHbetween 6 and 8 (e.g. pH 7), and optionally retains at least 95% of theoriginal peptide after 24 hours at 25° C. Yet another exemplary glucagonco-agonist analog exhibits about 10% or less of the activity of nativeglucagon at the glucagon receptor and at least about 20% of the activityof native GLP-1 at the GLP-1 receptor, is optionally soluble at aconcentration of at least 1 mg/mL at a pH between 6 and 8 (e.g. pH 7),and optionally retains at least 95% of the original peptide after 24hours at 25° C. Yet another exemplary glucagon co-agonist analogexhibits about 10% or less but above 0.1%, 0.5% or 1% of the activity ofnative glucagon at the glucagon receptor and at least about 50%, 60%,70%, 80%, 90% or 100% or more of the activity of native GLP-1 at theGLP-1 receptor, is optionally soluble at a concentration of at least 1mg/mL at a pH between 6 and 8 (e.g. pH 7), and optionally retains atleast 95% of the original peptide after 24 hours at 25° C. In someembodiments, such glucagon analogs retain at least 22, 23, 24, 25, 26,27 or 28 of the naturally occurring amino acids at the correspondingpositions in native glucagon (e.g. have 1-7, 1-5 or 1-3 modificationsrelative to naturally occurring glucagon).

Any one of the following peptides is excluded from the compounds of theinvention, although further modifications thereto exhibiting the desiredco-agonist activity, pharmaceutical compositions, kits, and treatmentmethods using such compounds may be included in the invention: Thepeptide of SEQ ID NO: 1 with an [Arg12] substitution and with aC-terminal amide; The peptide of SEQ ID NO: 1 with [Arg12,Lys20]substitutions and with a C-terminal amide; The peptide of SEQ ID NO: 1with [Arg12,Lys24] substitutions and with a C-terminal amide; Thepeptide of SEQ ID NO: 1 with [Arg12,Lys29] substitutions and with aC-terminal amide; The peptide of SEQ ID NO: 1 with a [Glu9]substitution; The peptide of SEQ ID NO: 1 missing His1, with [Glu9,Glu16, Lys29] substitutions and C-terminal amide; The peptide of SEQ IDNO: 1 with [Glu9, Glu16, Lys29] substitutions and with a C-terminalamide; The peptide of SEQ ID NO: 1 with [Lys13, Glu17] substitutionslinked via lactam bridge and with a C-terminal amide; The peptide of SEQID NO: 1 with [Lys17, Glu21] substitutions linked via lactam bridge andwith a C-terminal amide; The peptide of SEQ ID NO: 1 missing His1, with[Glu20, Lys24] substitutions linked via lactam bridge.

In accordance with one embodiment a pharmaceutical composition isprovided comprising any of the novel glucagon peptides disclosed herein,preferably sterile and preferably at a purity level of at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, and a pharmaceuticallyacceptable diluent, carrier or excipient. Such compositions may containa glucagon peptide at a concentration of at least 0.5 mg/ml, 1 mg/ml, 2mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml, 7 mg/ml, 8 mg/ml, 9 mg/ml, 10mg/ml, 11 mg/ml, 12 mg/ml, 13 mg/ml, 14 mg/ml, 15 mg/ml, 16 mg/ml, 17mg/ml, 18 mg/ml, 19 mg/ml, 20 mg/ml, 21 mg/ml, 22 mg/ml, 23 mg/ml, 24mg/ml, 25 mg/ml or higher. In one embodiment the pharmaceuticalcompositions comprise aqueous solutions that are sterilized andoptionally stored within various containers. The compounds of thepresent invention can be used in accordance with one embodiment toprepare pre-formulated solutions ready for injection. In otherembodiments the pharmaceutical compositions comprise a lyophilizedpowder. The pharmaceutical compositions can be further packaged as partof a kit that includes a disposable device for administering thecomposition to a patient. The containers or kits may be labeled forstorage at ambient room temperature or at refrigerated temperature.

In accordance with one embodiment a method of rapidly increasing glucoselevel or treating hypoglycemia using a pre-formulated aqueouscomposition of glucagon peptides of the invention is provided. Themethod comprises the step of administering an effective amount of anaqueous solution comprising a novel modified glucagon peptide of thepresent disclosure. In one embodiment the glucagon peptide is pegylatedat position 21 or 24 of the glucagon peptide and the PEG chain has amolecular weight of about 500 to about 5,000 Daltons. In one embodimentthe modified glucagon solution is prepackaged in a device that is usedto administer the composition to the patient suffering fromhypoglycemia.

In accordance with one embodiment an improved method of regulating bloodglucose levels in insulin dependent patients is provided. The methodcomprises the steps of administering insulin in an amounttherapeutically effective for the control of diabetes and administeringa novel modified glucagon peptide of the present disclosure in an amounttherapeutically effective for the prevention of hypoglycemia, whereinsaid administering steps are conducted within twelve hours of eachother. In one embodiment the glucagon peptide and the insulin areco-administered as a single composition, wherein the glucagon peptide ispegylated with a PEG chain having a molecular weight selected from therange of about 5,000 to about 40,000 Daltons

In another embodiment a method is provided for inducing the temporaryparalysis of the intestinal tract. The method comprises the step ofadministering one or more of the glucagon peptides disclosed herein to apatient.

In yet another embodiment a method of treating hyperglycemia, or amethod of reducing weight gain or inducing weight loss is provided,which involves administering an effective amount of an aqueous solutioncomprising a glucagon peptide of the invention. In one embodiment eithermethod comprises administering an effective amount of a compositioncomprising a glucagon agonist selected from the group consisting of SEQID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15,SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19. Inanother embodiment, the method comprises administering an effectiveamount of a composition comprising a glucagon agonist, wherein theglucagon agonist comprising a glucagon peptide selected from the groupconsisting of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:14, SEQ ED NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ IDNO: 19, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, and SEQ ID NO: 69,wherein amino acid 29 of the glucagon peptide is bound to a secondpeptide through a peptide bond, and said second peptide comprises thesequence of SEQ ID NO: 26, SEQ ID NO: 27 or SEQ ID NO: 28. In furtherembodiments, methods of treating diabetes involving co-administering aconventional dose or a reduced dose of insulin and a glucagon peptide ofthe invention are provided. Methods of treating diabetes with a glucagonpeptide of the invention, without co-administering insulin are alsoprovided.

In yet another aspect, the invention provides novel methods for treatinghyperglycemia and novel methods for decreasing appetite or promotingbody weight loss that involve administration of a glucagon/GLP-1co-agonist molecule (including pharmaceutically acceptable saltsthereof) that activates both the glucagon receptor and the GLP-1receptor. Agonism, i.e., activation, of both the glucagon and GLP-1receptors provides an unexpected improvement compared to GLP-1 agonismalone in treating hyperglycemia. Thus, the addition of glucagon agonismprovides an unexpected additive or synergistic effect, or otherunexpected clinical benefit(s). Administration with a conventional doseof insulin, a reduced dose of insulin, or without insulin iscontemplated according to such methods. Agonism of the glucagon receptoralso has an unexpected beneficial effect compared to GLP-1 agonism alonein promoting weight loss or preventing weight gain.

Exemplary glucagon/GLP-1 co-agonist molecules include glucagon peptidesof the invention, GLP-1 analogs that activate both GLP-1 and glucagonreceptors, fusions of glucagon and GLP-1, or fusions of glucagon analogsand GLP-1 analogs, or chemically modified derivatives thereof.Alternatively, a compound that activates the glucagon receptor can beco-administered with a compound that activates the GLP-1 receptor (suchas a GLP-1 analog, an exendin-4 analog, or derivatives thereof). Theinvention also contemplates co-administration of a glucagon agonistanalog with a GLP-1 agonist analog.

Such methods for treating hyperglycemia and/or for decreasing appetiteor promoting body weight loss include administration of a glucagonanalog with a modification at position 12 (e.g. Arg12), optionally incombination with modifications at position 16 and/or 20. The methods ofthe invention also include administration of glucagon analogs comprisingan intramolecular bridge between the side chains of two amino acidswithin the region of amino acids 12 and 29 that are separated by threeintervening amino acids, e.g. positions 12 and 16, positions 13 and 17(e.g., Lys13 Glu17 or Glu13 Lys17), positions 16 and 20, positions 17and 21 (e.g. Lys17 Glu 21 or Glu17 Lys 21), positions 20 and 24, orpositions 24 and 28, with the optional proviso that the amino acid atposition 9 is not Glu, and optionally including a C-terminal amide orester.

In accordance with one embodiment excluded from such glucagon/GLP-1co-agonist molecules are any glucagon analogs or GLP-1 analogs in theprior art known to be useful in such a method. In another embodimentpeptides described in U.S. Pat. No. 6,864,069 as acting as both a GLP-1agonist and a glucagon antagonist for treating diabetes are alsoexcluded as glucagon/GLP-1 co-agonist molecules. In another embodiment,excluded is the use of glucagon antagonists to treat diabetes, such asthe antagonists described in Unson et al., J. Biol. Chem., 264:789-794(1989), Ahn et al., J. Med. Chem., 44:3109-3116 (2001), and Sapse etal., Mol. Med., 8(5):251-262 (2002). In a further embodimentoxyntomodulin or a glucagon analog that contains the 8 C-terminal aminoacids of oxyntomodulin (SEQ ID NO: 27) are also excluded asglucagon/GLP-1 co-agonist molecules.

Such methods for treating hyperglycemia are expected to be useful for avariety of types of hyperglycemia, including diabetes, diabetes mellitustype I, diabetes mellitus type II, or gestational diabetes, eitherinsulin-dependent or non-insulin-dependent, and reducing complicationsof diabetes including nephropathy, retinopathy and vascular disease.Such methods for reducing appetite or promoting loss of body weight areexpected to be useful in reducing body weight, preventing weight gain,or treating obesity of various causes, including drug-induced obesity,and reducing complications associated with obesity including vasculardisease (coronary artery disease, stroke, peripheral vascular disease,ischemia reperfusion, etc.), hypertension, onset of diabetes type II,hyperlipidemia and musculoskeletal diseases.

All therapeutic methods, pharmaceutical compositions, kits and othersimilar embodiments described herein contemplate that the use of theterm glucagon analogs includes all pharmaceutically acceptable salts oresters thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph representing the stability of GlucagonCys²¹maleimidoPEG_(5K) at 37° C. incubated for 24, 48, 72, 96, 144 and166 hours, respectively.

FIG. 2 represents data generated from HPLC analysis of GlucagonCys²¹maleimidoPEG_(SK) at pH 5 incubated at 37° C. for 24, 72 or 144hours, respectively.

FIG. 3 represents data showing receptor mediated cAMP induction byglucagon analogs. More particularly, FIG. 3A compares induction of theglucagon receptor by glucagon analogs E16, K20 , E15, E16 ▴, E16, K20▾, E15, E16

, E16

and Gluc-NH₂ ▪

FIGS. 4A and 4B represents data showing receptor mediated cAMP inductionby glucagon analogs. More particularly, FIG. 4A compares induction ofthe glucagon receptor by glucagon analogs Gluc-NH₂ , E16Gluc-NH₂ ▴, E3,E16 Gluc-NH₂ ▾, Orn3, E16 Gluc-NH₂

and Nle3, E16 Gluc-NH₂,

relative to native glucagon ▪, whereas FIG. 4B compares induction of theGLP-1 receptor by glucagon analogs Gluc-NH₂ , E16 Gluc-NH₂ ▴, E3,E16Gluc-NH₂ ▾, Orn3, E16 Gluc-NH₂

and Nle3, E16 Gluc-NH₂,

relative to native GLP-1 ▪.

FIGS. 5A and 5B represents data showing receptor mediated cAMP inductionby glucagon analogs. More particularly, FIG. 5A compares induction ofthe glucagon receptor by glucagon analogs (E16, K20 Gluc-NH₂  (5 nM,stock solution), E15, E16 Gluc-NH₂ ▴ (5 nM, stock solution), E16, K20Gluc-NH₂ ▾ (10 nM, stock solution), E15, E16 Gluc-NH₂

(10 nM, stock solution) and E16 Gluc-NH₂

) relative to glucagon-NH₂ (▪), whereas FIG. 5B compares induction ofthe GLP-1 receptor by glucagon analogs (E16, K20 Gluc-NH₂ , E15, E16Gluc-NH₂ ▴, and E16 Gluc-NH₂,

) relative to GLP-1 (▪) and glucagon-NH₂ (□).

FIGS. 6A and 6B represents data showing receptor mediated cAMP inductionby glucagon analogs. More particularly, FIG. 6A compares induction ofthe glucagon receptor by glucagon analogs (Gluc-NH₂ , K12E16-NH₂ lactam▴, E16K20-NH₂ lactam ▾, K20E24-NH₂ lactam

and E24K28-NH₂ lactam

) relative to glucagon (▪), whereas FIG. 6B compares induction of theGLP-1 receptor by glucagon analogs (Gluc-NH₂ , K12E16-NH₂ lactam ▴,E16K20-NH₂ lactam ▾, K20E24-NH₂ lactam

and E24K28-NH₂ lactam

) relative to GLP-1 (▪).

FIGS. 7A and 7B represents data showing receptor mediated cAMP inductionby glucagon analogs. More particularly, FIG. 7A compares induction ofthe glucagon receptor by glucagon analogs (Gluc-NH₂ , E16 Gluc-NH₂, ▴,K12, E16 Gluc-NH₂ lactam ▾, E16, K20 Gluc-NH₂

and E16, K20 Gluc-NH₂ lactam

) relative to glucagon (▪), whereas FIG. 7B compares induction of theGLP-1 receptor by glucagon analogs (Gluc-NH₂ , E16 Gluc-NH₂, ▴, K12,E16 Gluc-NH₂ lactam ▾, E16, K20 Gluc-NH₂

and E16, K20 Gluc-NH₂ lactam

) relative to GLP-1 (▪).

FIGS. 8A-8F represent data showing receptor mediated cAMP induction byglucagon analogs at the glucagon receptor (FIGS. 8A, 8C and 8E) or theGLP-1 receptor (FIGS. 8B, 8C and 8F) wherein hE=homoglutamic acid andhC=homocysteic acid.

FIGS. 9A and 9B: represent data showing receptor mediated cAMP inductionby GLP (17-26) glucagon analogs, wherein amino acid positions 17-26 ofnative glucagon (SEQ ID NO: 1) have been substituted with the aminoacids of positions 17-26 of native GLP-1 (SEQ ID NO: 50). Moreparticularly, FIG. 9A compares induction of the glucagon receptor by thedesignated GLP (17-26) glucagon analogs, and FIG. 9B compares inductionof the GLP-1 receptor by the designated GLP (17-26) glucagon analogs.

FIGS. 10A-E: are graphs providing in vivo data demonstrating the abilityof the glucagon peptides of the present invention to induce weight lossin mice injected subcutaneously with the indicated amounts of therespective compounds. Sequence Identifiers for the glucagon peptidelisted in FIGS. 10A-10E are as follows, for FIG. 10A: Chimera 2 Aib2 C2440K PEG (SEQ ID NO: 486), Aib2 C24 Chimera 2 40K lactam (SEQ ID NO: 504)and Aib2 E16 K20 Gluc-NH2 Lac 40K (SEQ ID NO: 528); FIG. 10B: Aib2 C24Chi 2 lactam 40K (SEQ ID NO: 504), DMIA1 C24 Chi 2 Lactam 40K (SEQ IDNO: 505), Chimera 2 DMIA1 C24 40K (SEQ ID NO: 519), and Chimera 2 Aib2C24 40K (SEQ ID NO: 486), wherein the number at the end of the sequencedesignates the dosage used, either 70 or 350 nmol/kg; FIG. 10C: AIB2w/lactam C24 40K (SEQ ID NO: 504), AIB2 E16 K20 w/lactam C24 40K (SEQ IDNO: 528), DMIA1 E16 K20 w/lactam C24 40K (SEQ ID NO: 510), DMIA1 E16 K20w/lactam CEX 40K (SEQ ID NO: 513) and DMIA1 E16 K20 w/o lactam CEX 40K(SEQ ID NO: 529); FIG. 10D: AIB2 w lactam C24 40K (SEQ ID NO: 504), AIB2E16 K20 w lactam C24 40K (SEQ ID NO: 528), DMIA1 E16 K20 w lactam C2440K (SEQ ID NO: 510) and DMIA1 E16 K20 w lactam/Cex C24 40K (SEQ ID NO:513), wherein the number at the end of the sequence designates thedosage used, either 14 or 70 nmol/kg/wk; FIG. 10E: AIB2 w/o lactam C2440K (SEQ ID NO: 486), Chi 2 AIB2 C24 CEX 40K (SEQ ID NO: 533), AIB2 E16A18 K20 C24 40K (SEQ ID NO: 492), AIB2 w/o lactam CEX G29 C40 40K (SEQID NO: 488), AIB2 w/o lactam CEX C40 C41-2 (SEQ ID NO: 532), AIB2 w/olactam CEX C24 C40-2 (SEQ ID NO: 531) and AIB2 w/o lactam C24 60K (SEQID NO: 498), wherein the designation 40K or 60K represents the molecularweight of the polyethylene chain attached to the glucagon peptide.

DETAILED DESCRIPTION Definitions

In describing and claiming the invention, the following terminology willbe used in accordance with the definitions set forth below.

As used herein, the term “pharmaceutically acceptable carrier” includesany of the standard pharmaceutical carriers, such as a phosphatebuffered saline solution, water, emulsions such as an oil/water orwater/oil emulsion, and various types of wetting agents. The term alsoencompasses any of the agents approved by a regulatory agency of the USFederal government or listed in the US Pharmacopeia for use in animals,including humans.

As used herein the term “pharmaceutically acceptable salt” refers tosalts of compounds that retain the biological activity of the parentcompound, and which are not biologically or otherwise undesirable. Manyof the compounds disclosed herein are capable of forming acid and/orbase salts by virtue of the presence of amino and/or carboxyl groups orgroups similar thereto.

Pharmaceutically acceptable base addition salts can be prepared frominorganic and organic bases. Salts derived from inorganic bases, includeby way of example only, sodium, potassium, lithium, ammonium, calciumand magnesium salts. Salts derived from organic bases include, but arenot limited to, salts of primary, secondary and tertiary amines.

Pharmaceutically acceptable acid addition salts may be prepared frominorganic and organic acids. Salts derived from inorganic acids includehydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like. Salts derived from organic acids includeacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid,malic acid, malonic acid, succinic acid, maleic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluene-sulfonic acid,salicylic acid, and the like.

As used herein, the term “treating” includes prophylaxis of the specificdisorder or condition, or alleviation of the symptoms associated with aspecific disorder or condition and/or preventing or eliminating saidsymptoms. For example, as used herein the term “treating diabetes” willrefer in general to altering glucose blood levels in the direction ofnormal levels and may include increasing or decreasing blood glucoselevels depending on a given situation.

As used herein an “effective” amount or a “therapeutically effectiveamount” of a glucagon peptide refers to a nontoxic but sufficient amountof the peptide to provide the desired effect. For example one desiredeffect would be the prevention or treatment of hypoglycemia, asmeasured, for example, by an increase in blood glucose level. Analternative desired effect for the co-agonist analogs of the presentdisclosure would include treating hyperglycemia, e.g., as measured by achange in blood glucose level closer to normal, or inducing weightloss/preventing weight gain, e.g., as measured by reduction in bodyweight, or preventing or reducing an increase in body weight, ornormalizing body fat distribution. The amount that is “effective” willvary from subject to subject, depending on the age and general conditionof the individual, mode of administration, and the like. Thus, it is notalways possible to specify an exact “effective amount.” However, anappropriate “effective” amount in any individual case may be determinedby one of ordinary skill in the art using routine experimentation.

The term, “parenteral” means not through the alimentary canal but bysome other route such as subcutaneous, intramuscular, intraspinal, orintravenous.

As used herein, the term “purified” and like terms relate to theisolation of a molecule or compound in a form that is substantially freeof contaminants normally associated with the molecule or compound in anative or natural environment. As used herein, the term “purified” doesnot require absolute purity; rather, it is intended as a relativedefinition. The term “purified polypeptide” is used herein to describe apolypeptide which has been separated from other compounds including, butnot limited to nucleic acid molecules, lipids and carbohydrates.

The term “isolated” requires that the referenced material be removedfrom its original environment (e.g., the natural environment if it isnaturally occurring). For example, a naturally-occurring polynucleotidepresent in a living animal is not isolated, but the same polynucleotide,separated from some or all of the coexisting materials in the naturalsystem, is isolated.

As used herein, the term “peptide” encompasses a sequence of 3 or moreamino acids and typically less than 50 amino acids, wherein the aminoacids are naturally occurring or non-naturally occurring amino acids.Non-naturally occurring amino acids refer to amino acids that do notnaturally occur in vivo but which, nevertheless, can be incorporatedinto the peptide structures described herein.

As used herein, the terms “polypeptide” and “protein” are terms that areused interchangeably to refer to a polymer of amino acids, withoutregard to the length of the polymer. Typically, polypeptides andproteins have a polymer length that is greater than that of “peptides.”

A “glucagon peptide” as used herein includes any peptide comprising,either the amino acid sequence of SEQ ID NO: 1, or any analog of theamino acid sequence of SEQ ID NO: 1, including amino acid substitutions,additions, deletions or post translational modifications (e.g.,methylation, acylation, ubiquitination, intramolecular covalent bondingsuch as lactam bridge formation, PEGylation, and the like) of thepeptide, wherein the analog stimulates glucagon or GLP-1 receptoractivity, e.g., as measured by cAMP production using the assay describedin Example 14.

The term “glucagon agonist” refers to a complex comprising a glucagonpeptide that stimulates glucagon receptor activity, e.g., as measured bycAMP production using the assay described in Example 14.

As used herein a “glucagon agonist analog” is a glucagon peptidecomprising a sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQID NO: 15, or an analog of such a sequence that has been modified toinclude one or more conservative amino acid substitutions at one or moreof positions 2, 5, 7, 10, 11, 12, 13, 14, 17, 18, 19, 20, 21, 24, 27, 28or 29.

As used herein an amino acid “modification” refers to a substitution,addition or deletion of an amino acid, and includes substitution with oraddition of any of the 20 amino acids commonly found in human proteins,as well as atypical or non-naturally occurring amino acids. Throughoutthe application, all references to a particular amino acid position bynumber (e.g. position 28) refer to the amino acid at that position innative glucagon (SEQ ID NO:1) or the corresponding amino acid positionin any analogs thereof. For example, a reference herein to “position 28”would mean the corresponding position 27 for a glucagon analog in whichthe first amino acid of SEQ ID NO: 1 has been deleted. Similarly, areference herein to “position 28” would mean the corresponding position29 for a glucagon analog in which one amino acid has been added beforethe N-terminus of SEQ ID NO: 1. Commercial sources of atypical aminoacids include Sigma-Aldrich (Milwaukee, Wis.), ChemPep Inc. (Miami,Fla.), and Genzyme Pharmaceuticals (Cambridge, Mass.). Atypical aminoacids may be purchased from commercial suppliers, synthesized de novo,or chemically modified or derivatized from other amino acids.

As used herein a “glucagon co-agonist” is a glucagon peptide thatexhibits activity at the glucagon receptor of at least about 10% toabout 500% or more relative to native glucagon and also exhibitsactivity at the GLP-1 receptor of about at least 10% to about 200% ormore relative to native GLP-1.

As used herein a “glucagon/GLP-1 co-agonist molecule” is a molecule thatexhibits activity at the glucagon receptor of at least about 10%relative to native glucagon and also exhibits activity at the GLP-1receptor of at least about 10% relative to native GLP-1.

As used herein the term “native glucagon” refers to a peptide consistingof the sequence of SEQ ID NO: 1, and the term “native GLP-1” is ageneric term that designates GLP-1(7-36)amide (consisting of thesequence of SEQ ID NO: 52), GLP-1(7-37) acid (consisting of the sequenceof SEQ ID NO: 50) or a mixture of those two compounds. As used herein, ageneral reference to “glucagon” or “GLP-1” in the absence of any furtherdesignation is intended to mean native glucagon or native GLP-1,respectively.

As used herein an amino acid “substitution” refers to the replacement ofone amino acid residue by a different amino acid residue.

As used herein, the term “conservative amino acid substitution” isdefined herein as exchanges within one of the following five groups:

I. Small aliphatic, nonpolar or slightly polar residues:

-   -   Ala, Ser, Thr, Pro, Gly;

II. Polar, negatively charged residues and their amides and esters:

-   -   Asp, Asn, Glu, Gln, cysteic acid and homocysteic acid;

III. Polar, positively charged residues:

-   -   His, Arg, Lys; Ornithine (Orn)

IV. Large, aliphatic, nonpolar residues:

-   -   Met, Leu, Ile, Val, Cys, Norleucine (Nle), homocysteine

V. Large, aromatic residues:

-   -   Phe, Tyr, Trp, acetyl phenylalanine

As used herein the general term “polyethylene glycol chain” or “PEGchain”, refers to mixtures of condensation polymers of ethylene oxideand water, in a branched or straight chain, represented by the generalformula H(OCH₂CH₂)_(n)OH, wherein n is at least 9. Absent any furthercharacterization, the term is intended to include polymers of ethyleneglycol with an average total molecular weight selected from the range of500 to 40,000 Daltons. “polyethylene glycol chain” or “PEG chain” isused in combination with a numeric suffix to indicate the approximateaverage molecular weight thereof. For example, PEG-5,000 refers topolyethylene glycol chain having a total molecular weight average ofabout 5,000.

As used herein the term “pegylated” and like terms refers to a compoundthat has been modified from its native state by linking a polyethyleneglycol chain to the compound. A “pegylated glucagon peptide” is aglucagon peptide that has a PEG chain covalently bound to the glucagonpeptide:

As used herein a general reference to a peptide is intended to encompasspeptides that have modified amino and carboxy termini. For example, anamino acid chain comprising an amide group in place of the terminalcarboxylic acid is intended to be encompassed by an amino acid sequencedesignating the standard amino acids.

As used herein a “linker” is a bond, molecule or group of molecules thatbinds two separate entities to one another. Linkers may provide foroptimal spacing of the two entities or may further supply a labilelinkage that allows the two entities to be separated from each other.Labile linkages include photocleavable groups, acid-labile moieties,base-labile moieties and enzyme-cleavable groups.

As used herein a “dimer” is a complex comprising two subunits covalentlybound to one another via a linker. The term dimer, when used absent anyqualifying language, encompasses both homodimers and heterodimers. Ahomodimer comprises two identical subunits, whereas a heterodimercomprises two subunits that differ, although the two subunits aresubstantially similar to one another.

As used herein the term “charged amino acid” refers to an amino acidthat comprises a side chain that is negatively charged (i.e.,de-protonated) or positively charged (i.e., protonated) in aqueoussolution at physiological pH. For example negatively charged amino acidsinclude aspartic acid, glutamic acid, cysteic acid, homocysteic acid,and homoglutamic acid, whereas positively charged amino acids includearginine, lysine and histidine. Charged amino acids include the chargedamino acids among the 20 amino acids commonly found in human proteins,as well as atypical or non-naturally occurring amino acids.

As used herein the term “acidic amino acid” refers to an amino acid thatcomprises a second acidic moiety, including for example, a carboxylicacid or sulfonic acid group.

EMBODIMENTS

The invention provides glucagon peptides with increased or decreasedactivity at the glucagon receptor, or GLP-1 receptor, or both. Theinvention also provides glucagon peptides with altered selectivity forthe glucagon receptor versus the GLP-1 receptor.

Increased activity at the glucagon receptor is provided by an amino acidmodification at position 16 of native glucagon (SEQ ID NO: 1) asdescribed herein.

Reduced activity at the glucagon receptor is provided, e.g., by an aminoacid modification at position 3 as described herein.

Increased activity at the GLP-1 receptor is provided by replacing thecarboxylic acid of the C-terminal amino acid with a charge-neutralgroup, such as an amide or ester.

Increased activity at the GLP-1 receptor is provided by modificationsthat permit formation of an intramolecular bridge between the sidechains of two amino acids that are separated by three intervening aminoacids, for example, positions 12 and 16, or 16 and 20, or 20 and 24, asdescribed herein.

Increased activity at the GLP-1 receptor is provided by an amino acidmodification at position 20 as described herein.

Increased activity at the GLP-1 receptor is provided in glucagon analogscomprising the C-terminal extension of SEQ ID NO: 26. GLP-1 activity insuch analogs comprising SEQ ID NO: 26 can be further increased bymodifying the amino acid at position 18, 28 or 29, or at position 18 and29, as described herein.

Restoration of glucagon activity which has been reduced by amino acidmodifications at positions 1 and 2 is provided by a covalent bondbetween the side chains of two amino acids that are separated by threeintervening amino acids, for example, positions 12 and 16, or 16 and 20,or 20 and 24, as described herein.

A further modest increase in GLP-1 potency is provided by modifying theamino acid at position 10 to be Trp.

Any of the modifications described above which increase or decreaseglucagon receptor activity and which increase GLP-1 receptor activitycan be applied individually or in combination. Any of the modificationsdescribed above can also be combined with other modifications thatconfer other desirable properties, such as increased solubility and/orstability and/or duration of action. Alternatively, any of themodifications described above can be combined with other modificationsthat do not substantially affect solubility or stability or activity.Exemplary modifications include but are not limited to:

(A) Improving solubility, for example, by introducing one, two, three ormore charged amino acid(s) to the C-terminal portion of native glucagon,preferably at a position C-terminal to position 27. Such a charged aminoacid can be introduced by substituting a native amino acid with acharged amino acid, e.g. at positions 28 or 29, or alternatively byadding a charged amino acid, e.g. after position 27, 28 or 29. Inexemplary embodiments, one, two, three or all of the charged amino acidsare negatively charged. In other embodiments, one, two, three or all ofthe charged amino acids are positively charged. Such modificationsincrease solubility, e.g. provide at least 2-fold, 5-fold, 10-fold,15-fold, 25-fold, 30-fold or greater solubility relative to nativeglucagon at a given pH between about 5.5 and 8, e.g., pH 7, whenmeasured after 24 hours at 25° C.

(B) Increasing solubility and duration of action or half-life incirculation by addition of a hydrophilic moiety such as a polyethyleneglycol chain, as described herein, e.g. at position 16, 17, 20, 21, 24or 29, or at the C-terminus of the peptide.

(C) Increasing stability by modification of the aspartic acid atposition 15, for example, by deletion or substitution with glutamicacid, homoglutamic acid, cysteic acid or homocysteic acid. Suchmodifications can reduce degradation or cleavage at a pH within therange of 5.5 to 8, for example, retaining at least 75%, 80%, 90%, 95%,96%, 97%, 98% or 99% of the original peptide after 24 hours at 25° C.

(D) Increasing stability by modification of the amino acid at position27, for example, by substitution with methionine, leucine or norleucine.Such modifications can reduce oxidative degradation.

(E) Increasing resistance to dipeptidyl peptidase IV (DPP IV) cleavageby modification of the amino acid at position 1 or 2 as describedherein.

(F) Conservative substitutions, additions or deletions that do notaffect activity, for example, conservative substitutions at one or moreof positions 2, 5, 7, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, 24,27, 28 or 29; or deletion of amino acid 29 optionally combined with aC-terminal amide or ester in place of the C-terminal carboxylic acidgroup;

(G) Adding C-terminal extensions as described herein;

(H) Homodimerization or heterodimerization as described herein.

In exemplary embodiments, the glucagon peptide may comprise a total of1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to9, or up to 10 amino acid modifications relative to the native glucagonsequence.

One embodiment disclosed herein is directed to a glucagon agonist thathas been modified relative to the wild type peptide ofHis-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr(SEQ ID NO: 1) to enhance the peptide's potency at the glucagonreceptor. Surprisingly, applicants have discovered that the normallyoccurring serine at position 16 of native glucagon (SEQ ID NO: 1) can besubstituted with select acidic amino acids to enhance the potency ofglucagon, in tennis of its ability to stimulate cAMP synthesis in avalidated in vitro model assay (see Example 14). More particularly, thissubstitution enhances the potency of the analog at least 2-fold, 4-fold,5-fold, and up to 10-fold greater at the glucagon receptor. Thissubstitution also enhances the analog's activity at the GLP-1 receptorat least 5-fold, 10-fold, or 15-fold relative to native glucagon, butselectivity is maintained for the glucagon receptor over the GLP-1receptor.

In accordance with one embodiment the serine residue at position 16 ofnative glucagon is substituted with an amino acid selected from thegroup consisting of glutamic acid, glutamine, homoglutamic acid,homocysteic acid, threonine or glycine. In accordance with oneembodiment the serine residue at position 16 of native glucagon issubstituted with an amino acid selected from the group consisting ofglutamic acid, glutamine, homoglutamic acid and homocysteic acid, and inone embodiment the serine residue is substituted with glutamic acid. Inone embodiment the glucagon peptide having enhanced specificity for theglucagon receptor comprises the peptide of SEQ ID NO: 8, SEQ ID NO: 9,SEQ ID NO: 10 or a glucagon agonist analog thereof, wherein the carboxyterminal amino acid retains its native carboxylic acid group. Inaccordance with one embodiment a glucagon agonist comprising thesequence ofNH₂-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-COOH(SEQ ID NO: 10) is provided, wherein the peptide exhibits approximatelyfivefold enhanced potency at the glucagon receptor, relative to nativeglucagon as measured by the in vitro cAMP assay of Example 14.

The glucagon peptides of the present invention can be further modifiedto improve the peptide's solubility and stability in aqueous solutionsat physiological pH, while retaining the high biological activityrelative to native glucagon. In accordance with one embodiment,introduction of hydrophilic groups at positions 17, 21, and 24 of thepeptide of SEQ ID NO: 9 or SEQ ID NO: 10 are anticipated to improve thesolubility and stability of the high potency glucagon analog insolutions having a physiological pH. Introduction of such groups alsoincreases duration of action, e.g. as measured by a prolonged half-lifein circulation. Suitable hydrophilic moieties include any water solublepolymers known in the art, including PEG, homo- or co-polymers of PEG, amonomethyl-substituted polymer of PEG (mPEG), or polyoxyethyleneglycerol (POG). In accordance with one embodiment the hydrophilic groupcomprises a polyethylene (PEG) chain. More particularly, in oneembodiment the glucagon peptide comprises the sequence of SEQ ID NO: 6or SEQ ID NO: 7 wherein a PEG chain is covalently linked to the sidechains of amino acids present at positions 21 and 24 of the glucagonpeptide and the carboxy terminal amino acid of the peptide has thecarboxylic acid group.

The present disclosure also encompasses other conjugates in whichglucagon peptides of the invention are linked, optionally via covalentbonding and optionally via a linker, to a conjugate. Linkage can beaccomplished by covalent chemical bonds, physical forces suchelectrostatic, hydrogen, ionic, van der Waals, or hydrophobic orhydrophilic interactions. A variety of non-covalent coupling systems maybe used, including biotin-avidin, ligand/receptor, enzyme/substrate,nucleic acid/nucleic acid binding protein, lipid/lipid binding protein,cellular adhesion molecule partners; or any binding partners orfragments thereof which have affinity for each other.

Exemplary conjugates include but are not limited to a heterologouspeptide or polypeptide (including for example, a plasma protein), atargeting agent, an immunoglobulin or portion thereof (e.g. variableregion, CDR, or Fc region), a diagnostic label such as a radioisotope,fluorophore or enzymatic label, a polymer including water solublepolymers, or other therapeutic or diagnostic agents. In one embodiment aconjugate is provided comprising a glucagon peptide of the presentinvention and a plasma protein, wherein the plasma protein is selectedform the group consisting of albumin, transferin, fibrinogen andglubulins. In one embodiment the plasma protein moiety of the conjugateis albumin or transferin. In some embodiments, the linker comprises achain of atoms from 1 to about 60, or 1 to 30 atoms or longer, 2 to 5atoms, 2 to 10 atoms, 5 to 10 atoms, or 10 to 20 atoms long. In someembodiments, the chain atoms are all carbon atoms. In some embodiments,the chain atoms in the backbone of the linker are selected from thegroup consisting of C, O, N, and S. Chain atoms and linkers may beselected according to their expected solubility (hydrophilicity) so asto provide a more soluble conjugate. In some embodiments, the linkerprovides a functional group that is subject to cleavage by an enzyme orother catalyst or hydrolytic conditions found in the target tissue ororgan or cell. In some embodiments, the length of the linker is longenough to reduce the potential for steric hindrance. If the linker is acovalent bond or a peptidyl bond and the conjugate is a polypeptide, theentire conjugate can be a fusion protein. Such peptidyl linkers may beany length. Exemplary linkers are from about 1 to 50 amino acids inlength, 5 to 50, 3 to 5, 5 to 10, 5 to 15, or 10 to 30 amino acids inlength. Such fusion proteins may alternatively be produced byrecombinant genetic engineering methods known to one of ordinary skillin the art.

The present disclosure also encompasses glucagon fusion peptides orproteins wherein a second peptide or polypeptide has been fused to aterminus, e.g., the carboxy terminus of the glucagon peptide. Moreparticularly, the fusion glucagon peptide may comprise a glucagonagonist of SEQ ID NO: 55, SEQ ID NO: 9 or SEQ ID NO: 10 furthercomprising an amino acid sequence of SEQ ID NO: 26 (GPSSGAPPPS), SEQ EDNO: 27 (KRNRNNIA) or SEQ ID NO: 28 (KRNR) linked to amino acid 29 of theglucagon peptide. In one embodiment the amino acid sequence of SEQ IDNO: 26 (GPSSGAPPPS), SEQ ID NO: 27 (KRNRNNIA) or SEQ ID NO: 28 (KRNR) isbound to amino acid 29 of the glucagon peptide through a peptide bond.Applicants have discovered that in glucagon fusion peptides comprisingthe C-terminal extension peptide of Exendin-4 (e.g., SEQ ID NO: 26 orSEQ ID NO: 29), substitution of the native threonine residue at position29 with glycine dramatically increases GLP-1 receptor activity. Thisamino acid substitution can be used in conjunction with othermodifications disclosed herein to enhance the affinity of the glucagonanalogs for the GLP-1 receptor. For example, the T29G substitution canbe combined with the S16E and N20K amino acid substitutions, optionallywith a lactam bridge between amino acids 16 and 20, and optionally withaddition of a PEG chain as described herein. In one embodiment aglucagon/GLP-1 receptor co-agonist is provided, comprising the sequenceof SEQ ID NO: 64. In one embodiment the glucagon peptide portion of theglucagon fusion peptide is selected from the group consisting of SEQ IDNO: 55, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID

NO: 4, and SEQ ID NO: 5 wherein a PEG chain, when present at positions17, 21, 24, or the C-terminal amino acid, or at both 21 and 24, isselected from the range of 500 to 40,000 Daltons. More particularly, inone embodiment the glucagon peptide segment is selected from the groupconsisting of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 63, wherein thePEG chain is selected from the range of 500 to 5,000. In one embodimentthe glucagon peptide is a fusion peptide comprising the sequence of SEQID NO: 55 and SEQ ID NO: 65 wherein the peptide of SEQ ID NO: 65 islinked to the carboxy terminus of SEQ ID NO: 55.

In accordance with one embodiment, an additional chemical modificationof the glucagon peptide of SEQ ID NO: 10 bestows increased GLP-1receptor potency to a point where the relative activity at the glucagonand GLP-1 receptors is virtually equivalent. Accordingly, in oneembodiment a glucagon/GLP-1 receptor co-agonist is provided wherein theterminal amino acid of the glucagon peptides of the present inventionhave an amide group in place of the carboxylic acid group that ispresent on the native amino acid. The relative activity of the glucagonanalog at the respective glucagon and GLP-1 receptors can be adjusted byfurther modifications to the glucagon peptide to produce analogsdemonstrating about 40% to about 500% or more of the activity of nativeglucagon at the glucagon receptor and about 20% to about 200% or more ofthe activity of native GLP-1 at the GLP-1 receptor, e.g. 50-fold,100-fold or more increase relative to the normal activity of glucagon atthe GLP-1 receptor

In a further embodiment glucagon analogs are provided that exhibitglucagon/GLP-1 receptor co-agonist activity wherein an intramolecularbridge is formed between two amino acid side chains to stabilize thethree dimensional structure of the carboxy terminus of the peptide. Moreparticularly, the side chains of the amino acid pairs 12 and 16, 16 and20, 20 and 24 or 24 and 28 are linked to one another and thus stabilizethe glucagon alpha helix. The two side chains can be linked to oneanother through hydrogen-bonding, ionic interactions, such as theformation of salt bridges, or by covalent bonds. In some embodiments,the size of the ring or linker is about 8 atoms, or about 7-9 atoms.

Examples of amino acid pairings that are capable of covalently bondingto form a seven-atom linking bridge include Orn-Glu (lactam ring);Lys-Asp (lactam); or Homoser-Homoglu (lactone). Examples of amino acidpairings that may form an eight-atom linker include Lys-Glu (lactam);Homolys-Asp (lactam); Orn-Homoglu (lactam); 4-aminoPhe-Asp (lactam); orTyr-Asp (lactone). Examples of amino acid pairings that may form anine-atom linker include Homolys-Glu (lactam); Lys-Homoglu (lactam);4-aminoPhe-Glu (lactam); or Tyr-Glu (lactone). Any of the side chains onthese amino acids may additionally be substituted with additionalchemical groups, so long as the three-dimensional structure of thealpha-helix is not disrupted. One of ordinary skill in the art canenvision alternative pairings or alternative amino acid analogs,including chemically modifiedderivatives, that would create astabilizing structure of similar size and desired effect. For example, ahomocysteine-homocysteine disulfide bridge is 6 atoms in length and maybe further modified to provide the desired effect. Even without covalentlinkage, the amino acid pairings described above or similar pairingsthat one of ordinary skill in the art can envision may also provideadded stability to the alpha-helix through non-covalent bonds, forexample, through foiniation of salt bridges or hydrogen-bondinginteractions.

Further exemplary embodiments include the following pairings, optionallywith a lactam bridge: Glu at position 12 with Lys at position 16; nativeLys at position 12 with Glu at position 16; Glu at position 16 with Lysat position 20; Lys at position 16 with Glu at position 20; Glu atposition 20 with Lys at position 24; Lys at position 20 with Glu atposition 24; Glu at position 24 with Lys at position 28; Lys at position24 with Glu at position 28.

In accordance with one embodiment a glucagon analog is provided thatexhibits glucagon/GLP-1 receptor co-agonist activity wherein the analogcomprises an amino acid sequence selected from the group consisting ofSEQ ID NO: 11, 47, 48 and 49. In one embodiment the side chains arecovalently bound to one another, and in one embodiment the two aminoacids are bound to one another to form a lactam ring. The size of thelactam ring can vary depending on the length of the amino acid sidechains, and in one embodiment the lactam is formed by linking the sidechains of a lysine amino acid to a glutamic acid side chain.

The order of the amide bond in the lactam ring can be reversed (e.g., alactam ring can be formed between the side chains of a Lys12 and a Glu16or alternatively between a Glu 12 and a Lys16). In accordance with oneembodiment a glucagon analog of SEQ ID NO: 45 is provided wherein atleast one lactam ring is formed between the side chains of an amino acidpair selected from the group consisting of amino acid pairs 12 and 16,16 and 20, 20 and 24 or 24 and 28. In one embodiment a glucagon/GLP-1receptor co-agonist is provided wherein the co-agonist comprises aglucagon peptide analog of SEQ ID NO: 20 wherein the peptide comprisesan intramolecular lactam bridge formed between amino acid positions 12and 16 or between amino acid positions 16 and 20. In one embodiment aglucagon/GLP-1 receptor co-agonist is provided comprising the sequenceof SEQ ID NO: 20, wherein an intramolecular lactam bridge is formedbetween amino acid positions 12 and 16, between amino acid positions 16and 20, or between amino acid positions 20 and 24 and the amino acid atposition 29 is glycine, wherein the sequence of SEQ ID NO: 29 is linkedto the C-terminal amino acid of SEQ ID NO: 20. In a further embodimentthe amino acid at position 28 is aspartic acid.

The solubility of the glucagon peptide of SEQ ID NO: 20 can be furtherimproved, for example, by introducing one, two, three or more chargedamino acid(s) to the C-terminal portion of glucagon peptide of SEQ IDNO: 20, preferably at a position C-terminal to position 27. Such acharged amino acid can be introduced by substituting a native amino acidwith a charged amino acid, e.g. at positions 28 or 29, or alternativelyby adding a charged amino acid, e.g. after position 27, 28 or 29. Inexemplary embodiments, one, two, three or all of the charged amino acidsare negatively charged. Alternatively, solubility can also be enhancedby covalently linking hydrophilic moieties, such as polyethylene glycol,to the peptide.

In accordance with one embodiment, a glucagon analog is providedcomprising the sequence of SEQ ID NO: 55, wherein said analog differsfrom SEQ ID NO: 55 by 1 to 3 amino acids, selected from positions 1, 2,3, 5, 7, 10, 11, 13, 14, 17, 18, 19, 21, 24, 27, 28, and 29, whereinsaid glucagon peptide exhibits at least 20% of the activity of nativeGLP-1 at the GLP-1 receptor.

In accordance with one embodiment a glucagon/GLP-1 receptor co-agonistis provided comprising the sequence:NH₂-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Xaa-Xaa-Arg-Arg-Ala-Xaa-Asp-Phe-Val-Xaa-Trp-Leu-Met-Xaa-Xaa-R(SEQ ID NO: 33) wherein the Xaa at position 15 is selected from thegroup of amino acids consisting of Asp, Glu, cysteic acid, homoglutamicacid and homocysteic acid, Xaa at position 16 is selected from the groupof amino acids consisting of Ser, Glu, Gln, homoglutamic acid andhomocysteic acid, the Xaa at position 20 is Gln or Lys, the Xaa atposition 24 is Gln or Glu, the Xaa at position 28 is Asn, Lys or anacidic amino acid, the Xaa at position 29 is Thr, Gly or an acidic aminoacid, and R is COOH or CONH₂, with the proviso that when position 16 isserine, position 20 is Lys, or alternatively when position 16 is serinethe position 24 is Glu and either position 20 or position 28 is Lys.

In one embodiment the glucagon/GLP-1 receptor co-agonist comprises thesequence of SEQ ID NO: 33 wherein the amino acid at position 28 isaspartic acid and the amino acid at position 29 is glutamic acid. Inanother embodiment the amino acid at position 28 is the nativeasparagine, the amino acid at position 29 is glycine and the amino acidsequence of SEQ ID NO: 29 or SEQ ID NO: 65 is covalently linked to thecarboxy terminus of SEQ ID NO: 33.

In one embodiment a co-agonist is provided comprising the sequence ofSEQ ID NO: 33 wherein an additional acidic amino acid added to thecarboxy terminus of the peptide. In a further embodiment the carboxyterminal amino acid of the glucagon analog has an amide in place of thecarboxylic acid group of the natural amino acid. In one embodiment theglucagon analog comprises a sequence selected from the group consistingof SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43 and SEQ IDNO: 44.

In accordance with one embodiment a glucagon peptide analog of SEQ IDNO: 33 is provided, wherein said analog differs from SEQ ID NO: 33 by 1to 3 amino acids, selected from positions 1, 2, 3, 5, 7, 10, 11, 13, 14,17, 18, 19, 21 and 27, with the proviso that when the amino acid atposition 16 is serine, either position 20 is lysine, or a lactam bridgeis formed between the amino acid at position 24 and either the aminoacid at position 20 or position 28. In accordance with one embodimentthe analog differs from SEQ ID NO: 33 by 1 to 3 amino acids selectedfrom positions 1, 2, 3, 21 and 27. In one embodiment the glucagonpeptide analog of SEQ ID NO: 33 differs from that sequence by 1 to 2amino acids, or in one embodiment by a single amino acid, selected formpositions 1, 2, 3, 5, 7, 10, 11, 13, 14, 17, 18, 19, 21 and 27, with theproviso that when the amino acid at position 16 is serine, eitherposition 20 is lysine, or a lactam bridge is formed between the aminoacid at position 24 and either the amino acid at position 20 or position28.

In accordance with another embodiment a relatively selective GLP-1receptor agonist is provided comprising the sequenceNH2-His-Ser-Xaa-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Xaa-Xaa-Arg-Arg-Ala-Xaa-Asp-Phe-Val-Xaa-Trp-Leu-Met-Xaa-Xaa-R(SEQ ID NO: 53) wherein the Xaa at position 3 is selected from the groupof amino acids consisting of Glu, Om or Nle, the Xaa at position 15 isselected from the group of amino acids consisting of Asp, Glu, cysteicacid, homoglutamic acid and homocysteic acid, Xaa at position 16 isselected from the group of amino acids consisting of Ser, Glu, Gln,homoglutamic acid and homocysteic acid, the Xaa at position 20 is Gln orLys, the Xaa at position 24 is Gln or Glu, the Xaa at position 28 isAsn, Lys or an acidic amino acid, the Xaa at position 29 is Thr, Gly oran acidic amino acid, and R is COOH, CONH₂, SEQ ID NO: 26 or SEQ ID NO:29, with the proviso that when position 16 is serine, position 20 isLys, or alternatively when position 16 is serine the position 24 is Gluand either position 20 or position 28 is Lys. In one embodiment theamino acid at position 3 is glutamic acid. In one embodiment the acidicamino acid substituted at position 28 and/or 29 is aspartic acid orglutamic acid. In one embodiment the glucagon peptide, including aco-agonist peptide, comprises the sequence of SEQ ID NO: 33 furthercomprising an additional acidic amino acid added to the carboxy terminusof the peptide. In a further embodiment the carboxy terminal amino acidof the glucagon analog has an amide in place of the carboxylic acidgroup of the natural amino acid.

In accordance with one embodiment a glucagon/GLP-1 receptor co-agonistis provided comprising a modified glucagon peptide selected from thegroup consisting of:

NH₂-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Xaa-Xaa-Arg-Arg-Ala-Xaa-Asp-Phe-Val-Xaa-Trp-Leu-Met-Xaa-Xaa-R (SEQ ID NO: 34), wherein the Xaa atposition 15 is selected from the group of amino acids consisting of Asp,Glu, cysteic acid, homoglutamic acid and homocysteic acid, Xaa atposition 16 is selected from the group of amino acids consisting of Ser,Glu, Gln, homoglutamic acid and homocysteic acid, the Xaa at position 20is Gln or Lys, the Xaa at position 24 is Gln or Glu and the Xaa atposition 28 is Asn, Asp or Lys, R is COOH or CONH₂, the Xaa at position29 is Thr or Gly, and R is COOH, CONH₂, SEQ ID NO: 26 or SEQ ID NO: 29,with the proviso that when position 16 is serine, position 20 is Lys, oralternatively when position 16 is serine the position 24 is Glu andeither position 20 or position 28 is Lys. In one embodiment R is CONH₂,the Xaa at position 15 is Asp, the Xaa at position 16 is selected fromthe group of amino acids consisting of Glu, Gln, homoglutamic acid andhomocysteic acid, the Xaas at positions 20 and 24 are each Gln the Xaaat position 28 is Asn or Asp and the Xaa at position 29 is Thr. In oneembodiment the Xaas at positions 15 and 16 are each Glu, the Xaas atpositions 20 and 24 are each Gln, the Xaa at position 28 is Asn or Asp,the Xaa at position 29 is Thr and R is CONH₂.

It has been reported that certain positions of the native glucagonpeptide can be modified while retaining at least some of the activity ofthe parent peptide. Accordingly, applicants anticipate that one or moreof the amino acids located at positions at positions 2, 5, 7, 10, 11,12, 13, 14, 17, 18, 19, 20, 21, 24, 27, 28 or 29 of the peptide of SEQID NO: 11 can be substituted with an amino acid different from thatpresent in the native glucagon peptide, and still retain activity at theglucagon receptor. In one embodiment the methionine residue present atposition 27 of the native peptide is changed to leucine or norleucine toprevent oxidative degradation of the peptide. In another embodiment theamino acid at position 20 is substituted with Lys, Arg, Orn orCitrullene and/or position 21 is substituted with Glu, homoglutamic acidor homocysteic acid.

In one embodiment a glucagon analog of SEQ ID NO: 20 is provided wherein1 to 6 amino acids, selected from positions 1, 2, 5, 7, 10, 11, 13, 14,17, 18, 19, 21, 27, 28 or 29 of the analog differ from the correspondingamino acid of SEQ ID NO: 1, with the proviso that when the amino acid atposition 16 is serine, position 20 is Lys, or alternatively whenposition 16 is serine the position 24 is Glu and either position 20 orposition 28 is Lys. In accordance with another embodiment a glucagonanalog of SEQ ID NO: 20 is provided wherein 1 to 3 amino acids selectedfrom positions 1, 2, 5, 7, 10, 11, 13, 14, 17, 18, 19, 20, 21, 27, 28 or29 of the analog differ from the corresponding amino acid of SEQ IDNO: 1. In another embodiment, a glucagon analog of SEQ ID NO: 8, SEQ IDNO: 9 or SEQ ID NO: 11 is provided wherein 1 to 2 amino acids selectedfrom positions 1, 2, 5, 7, 10, 11, 13, 14, 17, 18, 19, 20 or 21 of theanalog differ from the corresponding amino acid of SEQ ID NO: 1, and ina further embodiment the one to two differing amino acids representconservative amino acid substitutions relative to the amino acid presentin the native glucagon sequence (SEQ ID NO: 1). In one embodiment aglucagon peptide of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQID NO: 15 is provided wherein the glucagon peptide further comprisesone, two or three amino acid substitutions at positions selected frompositions 2, 5, 7, 10, 11, 13, 14, 17, 18, 19, 20, 21, 27 or 29. In oneembodiment the substitutions at positions 2, 5, 7, 10, 11, 13, 14, 16,17, 18, 19, 20, 21, 27 or 29 are conservative amino acid substitutions.

In accordance with one embodiment a glucagon/GLP-1 receptor co-agonistis provided comprising a variant of the sequence of SEQ ID NO 33,wherein 1 to 10 amino acids selected from positions 16, 17, 18, 20, 21,23, 24, 27, 28 and 29, respectively, of the variant differ from thecorresponding amino acid of SEQ ID NO: 1. In accordance with oneembodiment a variant of the sequence of SEQ ID NO 33 is provided whereinthe variant differs from SEQ ID NO: 33 by one or more amino acidsubstitutions selected from the group consisting of Gln17, Ala18, Glu21,Ile23, Ala24, Val27 and Gly29. In accordance with one embodiment aglucagon/GLP-1 receptor co-agonist is provided comprising variants ofthe sequence of SEQ ID NO 33, wherein 1 to 2 amino acids selected frompositions 17-26 of the variant differ from the corresponding amino acidof SEQ ID NO: 1. In accordance with one embodiment a variant of thesequence of SEQ ID NO 33 is provided wherein the variant differs fromSEQ ID NO: 33 by an amino acid substitution selected from the groupconsisting of Gln17, Ala18, Glu21, Ile23 and Ala24. In accordance withone embodiment a variant of the sequence of SEQ ID NO 33 is providedwherein the variant differs from SEQ ID NO: 33 by an amino acidsubstitution at position 18 wherein the substituted amino acid isselected from the group consisting of Ala, Ser, Thr, Pro and Gly. Inaccordance with one embodiment a variant of the sequence of SEQ ID NO 33is provided wherein the variant differs from SEQ ID NO: 33 by an aminoacid substitution of Ala at position 18. Such variations are encompassedby SEQ ID NO: 55. In another embodiment a glucagon/GLP-1 receptorco-agonist is provided comprising variants of the sequence of SEQ ID NO33, wherein 1 to 2 amino acids selected from positions 17-22 of thevariant differ from the corresponding amino acid of SEQ ID NO: 1, and ina further embodiment a variant of SEQ ID NO 33 is provided wherein thevariant differs from SEQ ID NO: 33 by for 2 amino acid substitutions atpositions 20 and 21. In accordance with one embodiment a glucagon/GLP-1receptor co-agonist is provided comprising the sequence:

NH2-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Xaa-Xaa-Arg-Arg-Ala-Xaa-Xaa-Phe-Val-Xaa-Trp-Leu-Met-Xaa-Xaa-R(SEQ ID NO: 51), wherein the Xaa at position 15 is Asp, Glu, cysteicacid, homoglutamic acid or homocysteic acid, the Xaa at position 16 isSer, Glu, Gln, homoglutamic acid or homocysteic acid, the Xaa atposition 20 is Gln, Lys, Arg, Orn or citrulline, the Xaa at position 21is Asp, Glu, homoglutamic acid or homocysteic acid, the Xaa at position24 is Gln or Glu, the Xaa at position 28 is Asn, Lys or an acidic aminoacid, the Xaa at position 29 is Thr or an acid amino acid and R is COOHor CONH₂. In one embodiment R is CONH₂. In accordance with oneembodiment a glucagon/GLP-1 receptor co-agonist is provided comprising avariant of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14,SEQ ID NO: 15, SEQ ID NO: 47, SEQ ID NO: 48 or SEQ ID NO: 49, whereinthe variant differs from said sequence by an amino acid substitution atposition 20. In one embodiment the amino acid substitution is selectedform the group consisting of Lys, Arg, Orn or citrulline for position20.

In one embodiment a glucagon agonist is provided comprising an analogpeptide of SEQ ID NO: 34 wherein the analog differs from SEQ ID NO: 34by having an amino acid other than serine at position 2. In oneembodiment the serine residue is substituted with aminoisobutyric acidor alanine, and in one embodiment the serine residue is substituted withaminoisobutyric acid. Such modifications suppresses cleavage bydipeptidyl peptidase IV while retaining the inherent potency of theparent compound (e.g. at least 75, 80, 85, 90, 95% or more of thepotentcy of the parent compound). In one embodiment the solubility ofthe analog is increased, for example, by introducing one, two, three ormore charged amino acid(s) to the C-terminal portion of native glucagon,preferably at a position C-terminal to position 27. In exemplaryembodiments, one, two, three or all of the charged amino acids arenegatively charged. In another embodiment the analog further comprisesan acidic amino acid substituted for the native amino acid at position28 or 29 or an acidic amino acid added to the carboxy terminus of thepeptide of SEQ ID NO: 34.

In one embodiment the glucagon analogs disclosed herein are furthermodified at position 1 or 2 to reduce susceptibility to cleavage bydipeptidyl peptidase IV. In one embodiment a glucagon analog of SEQ IDNO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQID NO: 15 is provided wherein the analog differs from the parentmolecule by a substitution at position 2 and exhibits reducedsusceptibility (i.e. resistance) to cleavage by dipeptidyl peptidase IV.More particularly, in one embodiment position 2 of the analog peptide issubstituted with an amino acid selected from the group consisting ofd-serine, alanine, valine, amino n-butyric acid, glycine, N-methylserine and aminoisobutyric acid. In one embodiment position 2 of theanalog peptide is substituted with an amino acid selected from the groupconsisting of d-serine, alanine, glycine, N-methyl serine andaminoisobutyric acid. In another embodiment position 2 of the analogpeptide is substituted with an amino acid selected from the groupconsisting of d-serine glycine, N-methyl serine and aminoisobutyricacid. In one embodiment the glucagon peptide comprises the sequence ofSEQ ID NO: 21 or SEQ ID NO: 22.

In one embodiment a glucagon analog of SEQ ID NO: 9, SEQ ID NO: 11, SEQID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15 is providedwherein the analog differs from the parent molecule by a substitution atposition 1 and exhibits reduced susceptibility (i.e. resistance) tocleavage by dipeptidyl peptidase IV. More particularly, position 1 ofthe analog peptide is substituted with an amino acid selected from thegroup consisting of d-histidine, alpha, alpha-dimethyl imidiazole aceticacid (DMIA), N-methyl histidine, alpha-methyl histidine, imidazoleacetic acid, desaminohistidine, hydroxyl-histidine, acetyl-histidine andhomo-histidine. In another embodiment a glucagon agonist is providedcomprising an analog peptide of SEQ ID NO: 34 wherein the analog differsfrom SEQ ID NO: 34 by having an amino acid other than histidine atposition 1. In one embodiment the solubility of the analog is increased,for example, by introducing one, two, three or more charged aminoacid(s) to the C-terminal portion of native glucagon, preferably at aposition C-terminal to position 27. In exemplary embodiments, one, two,three or all of the charged amino acids are negatively charged. Inanother embodiment the analog further comprises an acidic amino acidsubstituted for the native amino acid at position 28 or 29 or an acidicamino acid added to the carboxy terminus of the peptide of SEQ ID NO:34. In one embodiment the acidic amino acid is aspartic acid or glutamicacid.

In one embodiment the glucagon/GLP-1 receptor co-agonist comprises asequence of SEQ ID NO: 20 further comprising an additional carboxyterminal extension of one amino acid or a peptide selected from thegroup consisting of SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28. Inthe embodiment wherein a single amino acid is added to the carboxyterminus of SEQ ID NO: 20, the amino acid is typically selected from oneof the 20 common amino acids, and in one embodiment the additionalcarboxy terminus amino acid has an amide group in place of thecarboxylic acid of the native amino acid. In one embodiment theadditional amino acid is selected from the group consisting of glutamicacid, aspartic acid and glycine.

In an alternative embodiment a glucagon/GLP-1 receptor co-agonist isprovided wherein the peptide comprises at least one lactam ring formedbetween the side chain of a glutamic acid residue and a lysine residue,wherein the glutamic acid residue and a lysine residue are separated bythree amino acids. In one embodiment the carboxy terminal amino acid ofthe lactam bearing glucagon peptide has an amide group in place of thecarboxylic acid of the native amino acid. More particularly, in oneembodiment a glucagon and GLP-1 co-agonist is provided comprising amodified glucagon peptide selected from the group consisting of:

(SEQ ID NO: 66)NH₂-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Xaa-Xaa-R (SEQ ID NO: 67)NH₂-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Lys-Asp-Phe-Val-Gln-Trp-Leu-Met-Xaa-Xaa-R (SEQ ED NO: 68)NH₂-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Lys-Asp-Phe-Val-Glu-Trp-Leu-Met-Xaa-Xaa-R (SEQ ID NO: 69)NH₂-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Glu-Trp-Leu-Met-Lys-Xaa-R (SEQ ID NO: 16)NH₂-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Lys-Asp-Phe-Val-Glu-Trp-Leu-Met-Asn-Thr-R (SEQ ID NO: 17)NH₂-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Gln-Asp-Phe-Val-Glu-Trp-Leu-Met-Lys-Thr-R (SEQ ID NO: 18)NH₂-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Lys-Asp-Phe-Val-Glu-Trp-Leu-Met-Lys-Thr-Rwherein Xaa at position 28=Asp, or Asn, the Xaa at position 29 is Thr orGly, R is selected from the group consisting of COOH, CONH₂, glutamicacid, aspartic acid, glycine, SEQ ID NO: 26, SEQ ID NO: 27 and SEQ IDNO: 28, and a lactam bridge is formed between Lys at position 12 and Gluat position 16 for SEQ ID NO: 66, between Glu at position 16 and Lys atposition 20 for SEQ ID NO: 67, between Lys at position 20 and Glu atposition 24 for SEQ ID NO: 68, between Glu at position 24 and Lys atposition 28 for SEQ ID NO: 69, between Lys at position 12 and Glu atposition 16 and between Lys at position 20 and Glu at position 24 forSEQ ID NO: 16, between Lys at position 12 and Glu at position 16 andbetween Glu at position 24 and Lys at position 28 for SEQ ID NO: 17 andbetween Glu at position 16 and Lys at position 20 and between Glu atposition 24 and Lys at position 28 for SEQ ID NO: 18. In one embodimentR is selected from the group consisting of COOH, CONH₂, glutamic acid,aspartic acid, glycine, the amino acid at position 28 is Asn, and theamino acid at position 29 is threonine. In one embodiment R is CONH₂,the amino acid at position 28 is Asn and the amino acid at position 29is threonine. In another embodiment R is selected from the groupconsisting of SEQ ID NO: 26, SEQ ID NO: 29 and SEQ ID NO: 65 and theamino acid at position 29 is glycine.

In a further embodiment the glucagon/GLP-1 receptor co-agonist isselected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12, SEQID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17and SEQ ID NO: 18, wherein the peptide further comprises an additionalcarboxy terminal extension of one amino acid or a peptide selected fromthe group consisting of SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28.In one embodiment the terminal extension comprises the sequence of SEQID NO: 26, SEQ ID NO: 29 or SEQ ID NO: 65 and the glucagon peptidecomprises the sequence of SEQ ID NO: 55. In one embodiment theglucagon/GLP-1 receptor co-agonist comprises the sequence of SEQ ID NO:33 wherein the amino acid at position 16 is glutamic acid, the aminoacid at position 20 is lysine, the amino acid at position 28 isasparagine and the amino acid sequence of SEQ ID No: 26 or SEQ ID NO: 29is linked to the carboxy terminus of SEQ ID NO: 33.

In the embodiment wherein a single amino acid is added to the carboxyterminus of SEQ ID NO: 20, the amino acid is typically selected from oneof the 20 common amino acids, and in one embodiment the amino acid hasan amide group in place of the carboxylic acid of the native amino acid.In one embodiment the additional amino acid is selected from the groupconsisting of glutamic acid and aspartic acid and glycine. In theembodiments wherein the glucagon agonist analog further comprises acarboxy terminal extension, the carboxy terminal amino acid of theextension, in one embodiment, ends in an amide group or an ester grouprather than a carboxylic acid.

In another embodiment the glucagon/GLP-1 receptor co-agonist comprisesthe sequence:NH₂-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Glu-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Xaa-CONH₂(SEQ ID NO: 19), wherein the Xaa at position 30 represents any aminoacid. In one embodiment Xaa is selected from one of the 20 common aminoacids, and in one embodiment the amino acid is glutamic acid, asparticacid or glycine. The solubility of this peptide can be further improvedby covalently linking a PEG chain to the side chain of amino acid atposition 17, 21, 24 or 30 of SEQ ID NO: 19. In a further embodiment thepeptide comprises an additional carboxy terminal extension of a peptideselected from the group consisting of SEQ ID NO: 26, SEQ ID NO: 27 andSEQ ID NO: 28. In accordance with one embodiment the glucagon/GLP-1receptor co-agonist comprises the sequence of SEQ ID NO: 30, SEQ ID NO:31 and SEQ ID NO: 32.

Additional site specific modifications internal to the glucagon sequenceof SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ IDNO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 andSEQ ID NO: 64 can be made to yield a set of glucagon agonists thatpossess variable degrees of GLP-1 agonism. Accordingly, peptides thatpossess virtually identical in vitro potency at each receptor have beenprepared and characterized. Similarly, peptides with tenfold selectivelyenhanced potency at each of the two receptors have been identified andcharacterized. As noted above substitution of the serine residue atposition 16 with glutamic acid enhances the potency of native glucagonat both the Glucagon and GLP-1 receptors, but maintains approximately atenfold selectivity for the glucagon receptor. In addition bysubstituting the native glutamine at position 3 with glutamic acid (SEQID NO: 22) generates a glucagon analog that exhibits approximately atenfold selectivity for the GLP-1 receptor.

The solubility of the glucagon/GLP-1 co-agonist peptides can be furtherenhanced in aqueous solutions at physiological pH, while retaining thehigh biological activity relative to native glucagon by the introductionof hydrophilic groups at positions 16, 17, 21, and 24 of the peptide, orby the addition of a single modified amino acid (i.e. an amino acidmodified to comprise a hydrophilic group) at the carboxy terminus of theglucagon/GLP-1 co-agonist peptide. In accordance with one embodiment thehydrophilic group comprises a polyethylene (PEG) chain. Moreparticularly, in one embodiment the glucagon peptide comprises thesequence of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13,SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO:18 wherein a PEG chain is covalently linked to the side chain of anamino acids at position 16, 17, 21, 24, 29 or the C-terminal amino acidof the glucagon peptide, with the proviso that when the peptidecomprises SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13the polyethylene glycol chain is covalently bound to an amino acidresidue at position 17, 21 or 24, when the peptide comprises SEQ ID NO:14 or SEQ ID NO: 15 the polyethylene glycol chain is covalently bound toan amino acid residue at position 16, 17 or 21, and when the peptidecomprises SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 18 the polyethyleneglycol chain is covalently bound to an amino acid residue at position 17or 21.

In one embodiment the glucagon peptide comprises the sequence of SEQ IDNO: 11, SEQ ID NO: 12 or SEQ ID NO: 13, wherein a PEG chain iscovalently linked to the side chain of an amino acids at position 17,21, 24, or the C-terminal amino acid of the glucagon peptide, and thecarboxy terminal amino acid of the peptide has an amide group in placeof the carboxylic acid group of the native amino acid. In one embodimentthe glucagon/GLP-1 receptor co-agonist peptide comprises a sequenceselected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ED NO: 17, SEQ ID NO: 18and SEQ ID NO: 19, wherein a PEG chain is covalently linked to the sidechain of an amino acid at position 17, 21 or 24 of SEQ ID NO: 12, SEQ IDNO: 13 and SEQ ID NO: 19, or at position 16, 17 or 21 of SEQ ID NO: 14and SEQ ID NO: 15 or at position 17 or 21 of SEQ ID NO: 16, SEQ ID NO:17 and SEQ ID NO: 18 of the glucagon peptide. In another embodiment theglucagon/GLP-1 receptor co-agonist peptide comprises the sequence of SEQID NO: 11 or SEQ ID NO: 19, wherein a PEG chain is covalently linked tothe side chain of an amino acids at position 17, 21 or 24 or theC-terminal amino acid of the glucagon peptide.

In accordance with one embodiment, and subject to the provisolimitations described in the preceding paragraphs, the glucagonco-agonist peptide is modified to contain one or more amino acidsubstitution at positions 16, 17, 21, 24, or 29 or the C-terminal aminoacid, wherein the native amino acid is substituted with an amino acidhaving a side chain suitable for crosslinking with hydrophilic moieties,including for example, PEG. The native peptide can be substituted with anaturally occurring amino acid or a synthetic (non-naturally occurring)amino acid. Synthetic or non-naturally occurring amino acids refer toamino acids that do not naturally occur in vivo but which, nevertheless,can be incorporated into the peptide structures described herein.Alternatively, the amino acid having a side chain suitable forcrosslinking with hydrophilic moieties, including for example, PEG, canbe added to the carboxy terminus of any of the glucagon analogsdisclosed herein. In accordance with one embodiment an amino acidsubstitution is made in the glucagon/GLP-1 receptor co-agonist peptideat a position selected from the group consisting of 16, 17, 21, 24, or29 replacing the native amino acid with an amino acid selected from thegroup consisting of lysine, cysteine, ornithine, homocysteine and acetylphenylalanine, wherein the substituting amino acid further comprises aPEG chain covalently bound to the side chain of the amino acid. In oneembodiment a glucagon peptide selected form the group consisting of SEQID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16,SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19 is further modified tocomprise a PEG chain is covalently linked to the side chain of an aminoacid at position 17 or 21 of the glucagon peptide. In one embodiment thepegylated glucagon/GLP-1 receptor co-agonist further comprises thesequence of SEQ ID NO: 26, SEQ ID NO: 27 or SEQ ID NO: 29.

In another embodiment the glucagon peptide comprises the sequence of SEQID NO: 55 or SEQ ID NO: 56, further comprising a C-terminal extension ofSEQ ID NO: 26, SEQ ID NO: 29 or SEQ ID NO: 65 linked to the C-terminalamino acid of SEQ ID NO: 55 or SEQ ID NO: 56, and optionally furthercomprising a PEG chain covalently linked to the side chain of an aminoacids at position 17, 18, 21, 24 or 29 or the C-terminal amino acid ofthe peptide. In another embodiment the glucagon peptide comprises thesequence of SEQ ID NO: 55 or SEQ ID NO: 56, wherein a PEG chain iscovalently linked to the side chain of an amino acids at position 21 or24 of the glucagon peptide and the peptide further comprises aC-terminal extension of SEQ ID NO: 26, or SEQ ID NO: 29.

In another embodiment the glucagon peptide comprises the sequence of SEQID NO: 55, or SEQ ID NO: 33 or SEQ ID NO: 34, wherein an additionalamino acid is added to the carboxy terminus of SEQ ID NO: 33 or SEQ IDNO: 34, and a PEG chain is covalently linked to the side chain of theadded amino acid. In a further embodiment, the pegylated glucagon analogfurther comprises a C-terminal extension of SEQ ID NO: 26 or SEQ ID NO:29 linked to the C-terminal amino acid of SEQ ID NO: 33 or SEQ ID NO:34. In another embodiment the glucagon peptide comprises the sequence ofSEQ ID NO: 19, wherein a PEG chain is covalently linked to the sidechain of the amino acid at position 30 of the glucagon peptide and thepeptide further comprises a C-terminal extension of SEQ ID NO: 26 or SEQID NO: 29 linked to the C-terminal amino acid of SEQ ID NO: 19.

The polyethylene glycol chain may be in the form of a straight chain orit may be branched. In accordance with one embodiment the polyethyleneglycol chain has an average molecular weight selected from the range ofabout 500 to about 10,000 Daltons. In one embodiment the polyethyleneglycol chain has an average molecular weight selected from the range ofabout 1,000 to about 5,000 Daltons. In an alternative embodiment thepolyethylene glycol chain has an average molecular weight selected fromthe range of about 10,000 to about 20,000 Daltons. In accordance withone embodiment the pegylated glucagon peptide comprises two or morepolyethylene chains covalently bound to the glucagon peptide wherein thetotal molecular weight of the glucagon chains is about 1,000 to about5,000 Daltons. In one embodiment the pegylated glucagon agonistcomprises a peptide consisting of SEQ ID NO: 5 or a glucagon agonistanalog of SEQ ID NO: 5, wherein a PEG chain is covalently linked to theamino acid residue at position 21 and at position 24, and wherein thecombined molecular weight of the two PEG chains is about 1,000 to about5,000 Daltons.

As described in detail in the Examples, the glucagon agonists of thepresent invention have enhanced biophysical stability and aqueoussolubility while demonstrating enhanced bioactivity relative to thenative peptide. Accordingly, the glucagon agonists of the presentinvention are believed to be suitable for any use that has previouslybeen described for the native glucagon peptide. Accordingly, themodified glucagon peptides described herein can be used to treathypoglycemia or to increase blood glucose level, to induce temporaryparalysis of the gut for radiological uses, or treat other metabolicdiseases that result from low blood levels of glucagon. The glucagonpeptides described herein also are expected to be used to reduce ormaintain body weight, or to treat hyperglycemia, or to reduce bloodglucose level, or to normalize blood glucose level.

The glucagon peptides of the invention may be administered alone or incombination with other anti-diabetic or anti-obesity agents.Anti-diabetic agents known in the art or under investigation includeinsulin, sulfonylureas, such as tolbutamide (Orinase), acetohexamide(Dymelor), tolazamide (Tolinase), chlorpropamide (Diabinese), glipizide(Glucotrol), glyburide (Diabeta, Micronase, Glynase), glimepiride(Amaryl), or gliclazide (Diamicron); meglitinides, such as repaglinide(Prandin) or nateglinide (Starlix); biguanides such as metformin(Glucophage) or phenformin; thiazolidinediones such as rosiglitazone(Avandia), pioglitazone (Actos), or troglitazone (Rezulin), or otherPPARy inhibitors; alpha glucosidase inhibitors that inhibit carbohydratedigestion, such as miglitol (Glyset), acarbose (Precose/Glucobay);exenatide (Byetta) or pramlintide; Dipeptidyl peptidase-4 (DPP-4)inhibitors such as vildagliptin or sitagliptin; SGLT (sodium-dependentglucose transporter 1) inhibitors; or FBPase (fructose1,6-bisphosphatase) inhibitors.

Anti-obesity agents known in the art or under investigation includeappetite suppressants, including phenethylamine type stimulants,phenteiniine (optionally with fenfluramine or dexfenfluramine),diethylpropion (Tenuate®), phendimetrazine (Prelu-2®, Bontril®),benzphetamine (Didrex®), sibutramine (Meridia®, Reductil®); rimonabant(Acomplia®), other cannabinoid receptor antagonists; oxyntomodulin;fluoxetine hydrochloride (Prozac); Qnexa (topiramate and phentermine),Excalia (bupropion and zonisamide) or Contrave (bupropion andnaltrexone); or lipase inhibitors, similar to xenical (Orlistat) orCetilistat (also known as ATL-962), or GT 389-255.

One aspect of the present disclosure is directed to a pre-formulatedaqueous solution of the presently disclosed glucagon agonist for use intreating hypoglycemia. The improved stability and solubility of theagonist compositions described herein allow for the preparation ofpre-formulated aqueous solutions of glucagon for rapid administrationand treatment of hypoglycemia. In one embodiment a solution comprising apegylated glucagon agonist is provided for administration to a patientsuffering from hypoglycemia, wherein the total molecular weight of thePEG chains linked to the pegylated glucagon agonist is between about 500to about 5,000 Daltons. In one embodiment the pegylated glucagon agonistcomprises a peptide selected from the group consisting of SEQ ID NO: 23,SEQ ID NO: 24, and SEQ ID NO: 25, and glucagon agonist analogs of SEQ IDNO: 23, SEQ ID NO: 24, and SEQ ID NO: 25, or a pegylated lactamderivative of glucagon comprising the sequence of SEQ ID NO: 20, whereinthe side chain of an amino acid residue of said glucagon peptide iscovalently bound to the polyethylene glycol chain.

The method of treating hypoglycemia in accordance with the presentinvention comprises the steps of administering the presently disclosedglucagon agonists to a patient using any standard route ofadministration, including parenterally, such as intravenously,intraperitoneally, subcutaneously or intramuscularly, intrathecally,transdermally, rectally, orally, nasally or by inhalation. In oneembodiment the composition is administered subcutaneously orintramuscularly. In one embodiment, the composition is administeredparenterally and the glucagon composition is prepackaged in a syringe.

Surprisingly, applicants have discovered that pegylated glucagonpeptides can be prepared that retain the parent peptide's bioactivityand specificity. However, increasing the length of the PEG chain, orattaching multiple PEG chains to the peptide, such that the totalmolecular weight of the linked PEG is greater than 5,000 Daltons, beginsto delay the time action of the modified glucagon. In accordance withone embodiment, a glucagon peptide of SEQ ID NO: 23, SEQ ID NO: 24, andSEQ ID NO: 25, or a glucagon agonist analog thereof, or a pegylatedlactam derivative of glucagon comprising the sequence of SEQ ID NO: 20is provided wherein the peptide comprises one or more polyethyleneglycol chains, wherein the total molecular weight of the linked PEG isgreater than 5,000 Daltons, and in one embodiment is greater than 10,000Daltons, but less than 40,000 Daltons. Such modified glucagon peptideshave a delayed or prolonged time of activity but without loss of thebioactivity.

Accordingly, such compounds can be administered to extend the effect ofthe administered glucagon peptide.

Glucagon peptides that have been modified to be covalently bound to aPEG chain having a molecular weight of greater than 10,000 Daltons canbe administered in conjunction with insulin to buffer the actions ofinsulin and help to maintain stable blood glucose levels in diabetics.The modified glucagon peptides of the present disclosure can beco-administered with insulin as a single composition, simultaneouslyadministered as separate solutions, or alternatively, the insulin andthe modified glucagon peptide can be administered at different timerelative to one another. In one embodiment the composition comprisinginsulin and the composition comprising the modified glucagon peptide areadministered within 12 hours of one another. The exact ratio of themodified glucagon peptide relative to the administered insulin will bedependent in part on deteiniining the glucagon levels of the patient,and can be determined through routine experimentation.

In accordance with one embodiment a composition is provided comprisinginsulin and a modified glucagon peptide selected from the groupconsisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 andglucagon agonist analogs thereof, wherein the modified glucagon peptidefurther comprises a polyethylene glycol chain covalently bound to anamino acid side chain at position 17, 21, 24 or 21 and 24. In oneembodiment the composition is an aqueous solution comprising insulin andthe glucagon analog. In embodiments where the glucagon peptide comprisesthe sequence of SEQ ID NO: 24 or SEQ ID NO: 25 the PEG chain iscovalently bound at position 21 or 24 of the glucagon peptide. In oneembodiment the polyethylene glycol chain has a molecular weight of about10,000 to about 40,000.

In accordance with one embodiment the modified glucagon peptidesdisclosed herein are used to induce temporary paralysis of theintestinal tract. This method has utility for radiological purposes andcomprises the step of administering an effective amount of apharmaceutical composition comprising a pegylated glucagon peptide, aglucagon peptide comprising a c-terminal extension or a dimer of suchpeptides. In one embodiment the glucagon peptide comprises a sequenceselected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ IDNO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ IDNO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 SEQ IDNO: 14 and SEQ ID NO: 15. In one embodiment the glucagon peptide furthercomprises a PEG chain, of about 1,000 to 40,000 Daltons is covalentlybound to an amino acid residue at position 21 or 24. In one embodimentthe glucagon peptide is selected from the group consisting of SEQ ID NO:10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQID NO: 15. In one embodiment the PEG chain has a molecular weight ofabout 500 to about 5,000 Daltons.

In a further embodiment the composition used to induce temporaryparalysis of the intestinal tract comprises a first modified glucagonpeptide and a second modified glucagon peptide. The first modifiedpeptide comprises a sequence selected from the group consisting of SEQID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 25, optionally linked to a PEGchain of about 500 to about 5,000 Daltons, and the second peptidecomprises a sequence selected from the group consisting of SEQ ID NO:23, SEQ ID NO: 24 and SEQ ID NO: 25, covalently linked to a PEG chain ofabout 10,000 to about 40,000 Daltons. In this embodiment the PEG chainof each peptide is covalently bound to an amino acid residue at eitherposition 17, 21 or 24 of the respective peptide, and independent of oneanother.

Oxyntomodulin, a naturally occurring digestive hormone found in thesmall intestine, has been reported to cause weight loss whenadministered to rats or humans (see Diabetes 2005; 54:2390-2395).Oxyntomodulin is a 37 amino acid peptide that contains the 29 amino acidsequence of glucagon (i.e. SEQ ID NO: 1) followed by an 8 amino acidcarboxy terminal extension of SEQ ID NO: 27 (KRNRNNIA). Accordingly,applicants believe that the bioactivity of oxyntomodulin can be retained(i.e. appetite suppression and induced weight loss/weight maintenance),while improving the solubility and stability of the compound andimproving the pharmacokinetics, by substituting the glucagon peptideportion of oxyntomodulin with the modified glucagon peptides disclosedherein. In addition applicants also believe that a truncatedOxyntomodulin molecule comprising a glucagon peptide of the invention,having the terminal four amino acids of oxyntomodulin removed will alsobe effective in suppressing appetite and inducing weight loss/weightmaintenance.

Accordingly, the present invention also encompasses the modifiedglucagon peptides of the present invention that have a carboxy terminalextension of SEQ ID NO: 27 (KRNRNNIA) or SEQ ID NO: 28. These compoundscan be administered to individuals to induce weight loss or preventweight gain. In accordance with one embodiment a glucagon agonist analogof SEQ ID NO: 33 or SEQ ID NO: 20, further comprising the amino acidsequence of SEQ ID NO: 27 (KRNRNNIA) or SEQ ID NO: 28 linked to aminoacid 29 of the glucagon peptide, is administered to individuals toinduce weight loss or prevent weight gain. More particularly, theglucagon peptide comprises a sequence selected from the group consistingof SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13 SEQ ID NO: 14 and SEQ IDNO: 15, further comprising the amino acid sequence of SEQ ID NO: 27(KRNRNNIA) or SEQ ID NO: 28 linked to amino acid 29 of the glucagonpeptide.

Exendin-4, is a peptide made up of 39 amino acids. It is a powerfulstimulator of a receptor known as GLP-1. This peptide has also beenreported to suppress appetite and induce weight loss. Applicants havefound that the terminal sequence of Exendin-4 when added at the carboxyterminus of glucagon improves the solubility and stability of glucagonwithout compromising the bioactivity of glucagon. In one embodiment theterminal ten amino acids of Exendin-4 (i.e. the sequence of SEQ ID NO:26 (GPSSGAPPPS)) are linked to the carboxy terminus of a glucagonpeptide of the present disclosure. These fusion proteins are anticipatedto have pharmacological activity for suppressing appetite and inducingweight loss/weight maintenance. In accordance with one embodiment aglucagon agonist analog of SEQ ID NO: 33 or SEQ ID NO: 20, furthercomprising the amino acid sequence of SEQ ID NO: 26 (GPSSGAPPPS) or SEQID NO: 29 linked to amino acid 29 of the glucagon peptide, isadministered to individuals to induce weight loss or prevent weightgain. More particularly, the glucagon peptide comprises a sequenceselected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 12, SEQID NO: 13 SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17,SEQ ID NO: 18, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO:69, SEQ ID NO: 55 and SEQ ID NO: 56 further comprising the amino acidsequence of SEQ ID NO: 26 (GPSSGAPPPS) or SEQ ID NO: 29 linked to aminoacid 29 of the glucagon peptide. In one embodiment the administeredglucagon peptide analog comprises the sequence of SEQ ID NO: 64.

The present disclosure also encompasses multimers of the modifiedglucagon peptides disclosed herein. Two or more of the modified glucagonpeptides can be linked together using standard linking agents andprocedures known to those skilled in the art. For example, dimers can beformed between two modified glucagon peptides through the use ofbifunctional thiol crosslinkers and bi-functional amine crosslinkers,particularly for the glucagon peptides that have been substituted withcysteine, lysine ornithine, homocysteine or acetyl phenylalanineresidues (e.g. SEQ ID NO: 3 and SEQ ID NO: 4). The dimer can be ahomodimer or alternatively can be a heterodimer. In one embodiment thedimer comprises a homodimer of a glucagon fusion peptide wherein theglucagon peptide portion comprises SEQ ID NO: 11 or SEQ ID NO: 20 and anamino acid sequence of SEQ ID NO: 26 (GPSSGAPPPS), SEQ ID NO: 27(KRNRNNIA) or SEQ ID NO: 28 (KRNR) linked to amino acid 29 of theglucagon peptide. In another embodiment the dimer comprises a homodimerof a glucagon agonist analog of SEQ ID NO: 11, wherein the glucagonpeptide further comprises a polyethylene glycol chain covalently boundto position 21 or 24 of the glucagon peptide.

In accordance with one embodiment a dimer is provided comprising a firstglucagon peptide bound to a second glucagon peptide via a linker,wherein the first glucagon peptide comprises a peptide selected from thegroup consisting of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ IDNO: 11 and the second glucagon peptide comprises SEQ ID NO: 20. Inaccordance with another embodiment a dimer is provided comprising afirst glucagon peptide bound to a second glucagon peptide via a linker,wherein said first glucagon peptide comprises a sequence selected fromthe group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ IDNO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and thesecond glucagon peptide comprise SEQ ID NO: 11, and pharmaceuticallyacceptable salts of said glucagon polypeptides. In accordance withanother embodiment a dimer is provided comprising a first glucagonpeptide bound to a second glucagon peptide via a linker, wherein saidfirst glucagon peptide is selected from the group consisting of SEQ IDNO: 11, SEQ 1D NO: 12, SEQ ID NO: 13 SEQ ID NO: 14, SEQ ID NO: 15, SEQID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18 and the second glucagonpeptide is independently selected from the group consisting of SEQ IDNO: 11, SEQ ID NO: 12, SEQ ID NO: 13 SEQ ID NO: 14, SEQ ID NO: 15, SEQID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, and pharmaceuticallyacceptable salts of said glucagon polypeptides. In one embodiment thefirst glucagon peptide is selected from the group consisting of SEQ IDNO: 20 and the second glucagon peptide is independently selected fromthe group consisting of SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 11. Inone embodiment the dimer is foiuied between two peptides wherein eachpeptide comprises the amino acid sequence of SEQ ID NO: 11.

The modified glucagon peptides of the present invention can be providedin accordance with one embodiment as part of a kit. In one embodiment akit for administering a glucagon agonist to a patient in need thereof isprovided wherein the kit comprises a modified glucagon peptide selectedfrom the group consisting of 1) a glucagon peptide comprising thesequence of SEQ ID NO: 20, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO:11;2) a glucagon fusion peptide comprising a glucagon agonist analog of SEQID NO: 11, SEQ ID NO: 20 or SEQ ID NO: 55, and an amino acid sequence ofSEQ ID NO: 26 (GPSSGAPPPS), SEQ ID NO: 27 (KRNRNNIA) or SEQ ID NO: 28(KRNR) linked to amino acid 29 of the glucagon peptide; and 3) apegylated glucagon peptide of SEQ ID NO: 11 or SEQ ID NO: 51, furthercomprising an amino acid sequence of SEQ ID NO: 26 (GPSSGAPPPS), SEQ IDNO: 27 (KRNRNNIA) or SEQ ID NO: 28 (KRNR) linked to amino acid 29 of theglucagon peptide, wherein the PEG chain covalently bound to position 17,21 or 24 has a molecular weight of about 500 to about 40,000 Daltons. Inone embodiment the kit comprise a glucagon/GLP-1 co-agonist wherein thepeptide comprises a sequence selected from the group consisting of SEQID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 SEQ ID NO: 14, SEQ ID NO: 15,SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18.

In one embodiment the kit is provided with a device for administeringthe glucagon composition to a patient, e.g. syringe needle, pen device,jet injector or other needle-free injector. The kit may alternatively orin addition include one or more containers, e.g., vials, tubes, bottles,single or multi-chambered pre-filled syringes, cartridges, infusionpumps (external or implantable), jet injectors, pre-filled pen devicesand the like, optionally containing the glucagon peptide in alyophilized form or in an aqueous solution. Preferably, the kits willalso include instructions for use. In accordance with one embodiment thedevice of the kit is an aerosol dispensing device, wherein thecomposition is prepackaged within the aerosol device. In anotherembodiment the kit comprises a syringe and a needle, and in oneembodiment the sterile glucagon composition is prepackaged within thesyringe.

The compounds of this invention may be prepared by standard syntheticmethods, recombinant DNA techniques, or any other methods of preparingpeptides and fusion proteins. Although certain non-natural amino acidscannot be expressed by standard recombinant DNA techniques, techniquesfor their preparation are known in the art. Compounds of this inventionthat encompass non-peptide portions may be synthesized by standardorganic chemistry reactions, in addition to standard peptide chemistryreactions when applicable.

EXAMPLES

General Synthesis Protocol:

Glucagon analogs were synthesized using HBTU-activated “Fast Boc” singlecoupling starting from 0.2 mmole of Boc Thr(OBzl)Pam resin on a modifiedApplied Biosystem 430 A peptide synthesizer. Boc amino acids and HBTUwere obtained from Midwest Biotech (Fishers, Ind.). Side chainprotecting groups used were: Arg(Tos), Asn(Xan), Asp(OcHex),Cys(pMeBzl), His(Bom), Lys(2C1-Z), Ser(OBzl), Thr(OBzl), Tyr(2Br—Z), andTrp(CHO). The side-chain protecting group on the N-terminal His was Boc.

Each completed peptidyl resin was treated with a solution of 20%piperidine in dimethylformamide to remove the formyl group from thetryptophan. Liquid hydrogen fluoride cleavages were performed in thepresence of p-cresol and dimethyl sulfide. The cleavage was run for 1hour in an ice bath using an HF apparatus (Penninsula Labs). Afterevaporation of the HF, the residue was suspended in diethyl ether andthe solid materials were filtered. Each peptide was extracted into 30-70ml aqueous acetic acid and a diluted aliquot was analyzed by HPLC[Beckman System Gold, 0.46×5 cm Zorbax C8, 1 ml/min, 45C, 214 nm, Abuffer=0.1% TFA,

B=0.1% TFA/90% acetonitrile, gradient of 10% to 80% B over 10 min].

Purification was done on a FPLC over a 2.2×25 cm Kromasil C18 columnwhile monitoring the UV at 214 nm and collecting 5 minute fractions. Thehomogeneous fractions were combined and lyophilized to give a productpurity of >95%. The correct molecular mass and purity were confirmedusing MALDI-mass spectral analysis.

General Pegylation Protocol: (Cys-maleimido)

Typically, the glucagon Cys analog is dissolved in phosphate bufferedsaline (5-10 mg/ml) and 0.01-Methylenediamine tetraacetic acid is added(10-15% of total volume). Excess (2-fold) maleimido methoxyPEG reagent(Nektar) is added and the reaction stirred at room temp while monitoringreaction progress by HPLC. After 8-24 hrs, the reaction mixture, isacidified and loaded onto a preparative reverse phase column forpurification using 0.1% TFA/acetonitrile gradient. The appropriatefractions were combined and lyophilized to give the desired pegylatedanalogs.

Example 1 Synthesis of Glucagon Cys¹⁷(1-29) and Similar MonoCys Analogs

0.2 mmole Boc Thr(OBzl) Pam resin (SynChem Inc) in a 60 ml reactionvessel and the following sequence was entered and run on a modifiedApplied Biosystems 430A Peptide Synthesizer using FastBoc HBTU-activatedsingle couplings.

(SEQ ID NO: 35) HSQGTFTSDYSKYLDSCRAQDFVQWLMNTThe following side chain protecting groups were used: Arg(Tos),Asp(OcHex), Asn(Xan), Cys(pMeBzl), Glu(OcHex), His(Boc), Lys(2C1-Z),Ser(Bzl), Thr(Bzl), Trp(CHO), and Tyr(Br—Z). The completed peptidylresin was treated with 20% piperidine/dimethylformamide to remove theTrp formyl protection then transferred to an HF reaction vessel anddried in vacuo. 1.0 ml p-cresol and 0.5 ml dimethyl sulfide were addedalong with a magnetic stir bar. The vessel was attached to the HFapparatus (Pennisula Labs), cooled in a dry ice/methanol bath,evacuated, and aprox. 10 ml liquid hydrogen fluoride was condensed in.The reaction was stirred in an ice bath for 1 hr then the HF was removedin vacuo. The residue was suspended in ethyl ether; the solids werefiltered, washed with ether, and the peptide extracted into 50 mlaqueous acetic acid. An analytical HPLC was run [0.46×5 cm Zorbax C8, 1ml/min, 45C, 214 nm, A buffer of 0.1% TFA, B buffer of 0.1% TFA/90% ACN,gradient=10% B to 80% B over 10 min.] with a small sample of thecleavage extract. The remaining extract was loaded onto a 2.2×25 cmKromasil C18 preparative reverse phase column and an acetonitrilegradient was run using a Phaiunacia FPLC system. 5 min fractions werecollected while monitoring the UV at 214 nm (2.0 A). A=0.1% TFA,B=0.1% TFA/50% acetonitrile. Gradient=30% B to 100% B over 450 min.

The fractions containing the purest product (48-52) were combinedfrozen, and lyophilized to give 30.1 mg. An HPLC analysis of the productdemonstrated a purity of >90% and MALDI mass spectral analysisdemonstrated the desired mass of 3429.7. Glucagon Cys²¹, Glucagon Cys²⁴,and Glucagon Cys²⁹ were similarly prepared.

Example 2 Synthesis of Glucagon-Cex and Other C-Terminal ExtendedAnalogs

285 mg (0.2 mmole) methoxybenzhydrylamine resin (Midwest Biotech) wasplaced in a 60 ml reaction vessel and the following sequence was enteredand run on a modified Applied Biosystems 430A peptide synthesizer usingFastBoc HBTU-activated single couplings.

(SEQ ID NO: 36) HSQGTFTSDYSKYLDSRRAQDFVQWLMNTGPSSGAPPPSThe following side chain protecting groups were used: Arg(Tos),Asp(OcHex), Asn(Xan), Cys(pMeBzl), Glu(OcHex), His(Boc), Lys(2Cl—Z),Ser(Bzl), Thr(Bzl), Trp(CHO), and Tyr(Br—Z). The completed peptidylresin was treated with 20% piperidine/dimethylformamide to remove theTrp formyl protection then transferred to HF reaction vessel and driedin vacuo. 1.0 ml p-cresol and 0.5 ml dimethyl sulfide were added alongwith a magnetic stir bar. The vessel was attached to the HF apparatus(Pennisula Labs), cooled in a dry ice/methanol bath, evacuated, andaprox. 10 ml liquid hydrogen fluoride was condensed in. The reaction wasstirred in an ice bath for 1 hr then the HF was removed in vacuo. Theresidue was suspended in ethyl ether; the solids were filtered, washedwith ether, and the peptide extracted into 50 ml aqueous acetic acid. Ananalytical HPLC was run [0.46×5 cm Zorbax C8, 1 ml/min, 45C, 214 nm, Abuffer of 0.1% TFA, B buffer of 0.1% TFA/90% ACN, gradient=10% B to 80%B over 10 min.] on an aliquot of the cleavage extract. The extract wasloaded onto a 2.2×25 cm Kromasil C18 preparative reverse phase columnand an acetonitrile gradient was run for elution using a Pharmacia FPLCsystem. 5 min fractions were collected while monitoring the UV at 214 nm(2.0 A). A=0.1% TFA,B=0.1% TFA/50% acetonitrile. Gradient=30% B to 100% B over 450 min.Fractions 58-65 were combined, frozen and lyophilized to give 198.1 mg.

HPLC analysis of the product showed a purity of greater than 95%. MALDImass spectral analysis showed the presence of the desired theoreticalmass of 4316.7 with the product as a C-terminal amide. Oxyntomodulin andoxyntomodulin-KRNR were similarly prepared as the C-terminal carboxylicacids starting with the appropriately loaded PAM-resin.

Example 3 Glucagon Cys¹⁷ Mal-PEG-5K

15.1 mg of Glucagon Cys¹⁷(1-29) and 27.3 mg methoxy poly(ethyleneglycol)maleimide avg. M.W.5000 (mPEG-Mal-5000,Nektar Therapeutics) weredissolved in 3.5 ml phosphate buffered saline (PBS) and 0.5 ml0.01-Methylenediamine tetraacetic acid (EDTA) was added. The reactionwas stirred at room temperature and the progress of the reaction wasmonitored by HPLC analysis [0.46×5 cm Zorbax C8, 1 ml/min, 45C, 214 nm(0.5 A), A=0.1% TFA, B=0.1% TFA/90% ACN, gradient=10% B to 80% B over 10min.].

After 5 hours, the reaction mixture was loaded onto 2.2×25 cm KromasilC18 preparastive reverse phase column. An acetonitrile gradient was runon a Pharmacia FPLC while monitoring the UV wavelength at 214 nm andcollecting 5 min fractions. A=0.1% TFA, B=0.1% TFA/50% acetonitrile,gradient=30% B to 100% B over 450 min. The fractions corresponding tothe product were combined, frozen and lyophilized to give 25.9 mg.

This product was analyzed on HPLC [0.46×5 cm Zorbax C8, 1 ml/min, 45C,214 nm (0.5 A), A=0.1% TFA, B=0.1% TFA/90% ACN, gradient=10% B to 80% Bover 10 min.] which showed a purity of aprox. 90%. MALDI (matrixassisted laser desorption ionization) mass spectral analysis showed abroad mass range (typical of PEG derivatives) of 8700 to 9500. Thisshows an addition to the mass of the starting glucagon peptide (3429) ofapproximately 5,000 a.m.u.

Example 4 Glucagon Cys²¹ Mal-PEG-5K

21.6 mg of Glucagon Cys²¹(1-29) and 24 mg mPEG-MAL-5000 (NektarTherapeutics) were dissolved in 3.5 ml phosphate buffered saline (PBS)and 0.5 ml 0.01M ethylene diamine tetraacetic acid (EDTA) was added. Thereaction was stirred at room temp. After 2 hrs, another 12.7 mg ofmPEG-MAL-5000 was added. After 8 hrs, the reaction mixture was loadedonto a 2.2×25 cm Vydac C18 preparative reverse phase column and anacetonitrile gradient was run on a Pharmacia FPLC at 4 ml/min whilecollecting 5 min fractions. A=0.1% TFA, B=0.1% TFA/50% ACN. Gradient=20%to 80% B over 450 min.

The fractions corresponding to the appearance of product were combinedfrozen and lyophilized to give 34 mg. Analysis of the product byanalytical HPLC [0.46×5 cm Zorbax C8, 1 ml/min, 45C, 214 nm (0.5 A),A=0.1% TFA,

B=0.1% TFA/90% ACN, gradient=10% B to 80% B over 10 min.] showed ahomogeneous product that was different than starting glucagon peptide.MALDI (matrix assisted laser desorption ionization) mass spectralanalysis showed a broad mass range (typical of PEG analogs) of 8700 to9700. This shows an addition to the mass of the starting glucagonpeptide (3470) of approximately 5,000 a.m.u.

Example 5 Glucagon Cys²⁴ Mal-PEG-5K

20.1 mg Glucagon C²⁴(1-29) and 39.5 mg mPEG-Mal-5000 (NektarTherapeutics) were dissolved in 3.5 ml PBS with stirring and 0.5 ml0.01M EDTA was added. The reaction was stirred at room temp for 7 hrs,then another 40 mg of mPEG-Mal-5000 was added. After approximately 15hr, the reaction mixture was loaded onto a 2.2×25 cm Vydac C18preparative reverse phase column and an acetonitrile gradient was runusing a Pharmacia FPLC. 5 min. fractions were collected while monitoringthe UV at 214 nm (2.0 A). A buffer=0.1% TFA, B buffer=0.1% TFA/50% ACN,gradient=30% B to 100% B over 450 min. The fractions corresponding toproduct were combined, frozen and lyophilized to give 45.8 mg. MALDImass spectral analysis showed a typical PEG broad signal with a maximumat 9175.2 which is approximately 5,000 a.m.u. more than Glucagon C²⁴(3457.8).

Example 6 Glucagon Cys²⁴ Mal-PEG-20K

25.7 mg of Glucagon Cys²⁴(1-29) and 40.7 mg mPEG-Mal-20K (NektarTherapeutics) were dissolved in 3.5 ml PBS with stirring at room temp.and 0.5 ml 0.01M EDTA was added. After 6 hrs, the ratio of startingmaterial to product was aprox. 60:40 as determined by HPLC. Another 25.1mg of mPEG-Mal-20K was added and the reaction allowed to stir another 16hrs. The product ratio had not significantly improved, so the reactionmixture was loaded onto a 2.2×25 cm Kromasil C18 preparative reversephase column and purified on a Pharmacia FPLC using a gradient of 30% Bto 100% B over 450 min. A buffer=0.1% TFA, B buffer=0.1% TFA/50% ACN,flow=4 ml/min, and 5 min fractions were collected while monitoring theUV at 214 nm (2.0 A). The fractions containing homogeneous product werecombined, frozen and lyophilized to give 25.7 mg. Purity as determinedby analytical HPLC was ˜90%. A MALDI mass spectral analysis showed abroad peak from 23,000 to 27,000 which is approximately 20,000 a.m.u.more than starting Glucagon C²⁴ (3457.8).

Example 7 Glucagon Cys²⁹ Mal-PEG-5K

20.0 mg of Glucagon Cys²⁹(1-29) and 24.7 mg mPEG-Mal-5000 (NektarTherapeutics) were dissolved in 3.5 ml PBS with stirring at roomtemperature and 0.5 ml 0.01M EDTA was added. After 4 hr, another 15.6 mgof mPEG-Mal-5000 was added to drive the reaction to completion. After 8hrs, the reaction mixture was loaded onto a 2.2×25 cm Vydac C18preparative reverse phase column and an acetonitrile gradient was run ona Pharmacia FPLC system. 5 min fractions were collected while monitoringthe UV at 214 nm (2.0 A). A=0.1% TFA,

B=0.1% TFA/50% ACN. Fractions 75-97 were combined frozen and lyophilizedto give 40.0 mg of product that is different than recovered startingmaterial on HPLC (fractions 58-63). Analysis of the product byanalytical HPLC [0.46×5 cm Zorbax C8, 1 ml/min, 45C, 214 nm (0.5 A),A=0.1% TFA, B=0.1% TFA/90% ACN, gradient=10% B to 80% B over 10 min.]showed a purity greater than 95%. MALDI mass spectral analysis showedthe presence of a PEG component with a mass range of 8,000 to 10,000(maximum at 9025.3) which is 5,540 a.m.u. greater than starting material(3484.8).

Example 8 Glucagon Cys²⁴ (2-butyrolactone)

To 24.7 mg of Glucagon Cys²⁴(1-29) was added 4 ml 0.05M ammoniumbicarbonate/50% acetonitrile and 5.5 ul of a solution of2-bromo-4-hydroxybutyric acid-γ-lactone (100 ul in 900 ul acetonitrile).After 3 hrs of stirring at room temperature, another 105 ul of lactonesolution was added to the reaction mixture which was stirred another 15hrs. The reaction mixture was diluted to 10 ml with 10% aqueous aceticacid and was loaded onto a 2.2×25 cm Kromasil C18 preparative reversephase column. An acetonitrile gradient (20% B to 80% B over 450 min) wasrun on a Pharmacia FPLC while collecting 5 min fractions and monitoringthe UV at 214 nm (2.0 A). Flow=4 ml/min, A=0.1% TFA, B=0.1% TFA/50% ACN.Fractions 74-77 were combined frozen and lyophilized to give 7.5 mg.HPLC analysis showed a purity of 95% and MALDI mass spect analysisshowed a mass of 3540.7 or 84 mass units more than starting material.This result consistent with the addition of a single butyrolactonemoiety.

Example 9 Glucagon Cys²⁴(S-carboxymethyl)

18.1 mg of Glucagon Cys²⁴(1-29) was dissolved in 9.4 ml 0.1M sodiumphosphate buffer (pH=9.2) and 0.6 ml bromoacetic acid solution (1.3mg/ml in acetonitrile) was added. The reaction was stirred at roomtemperature and the reaction progress was followed by analytical HPLC.After 1 hr another 0.1 ml bromoacetic acid solution was added. Thereaction was stirred another 60 min. then acidified with aqueous aceticacid and was loaded onto a 2.2×25 cm Kromasil C18 preparative reversephase column for purification. An acetonitrile gradient was run on aPharmacia FPLC (flow=4 ml/min) while collecting 5 min fractions andmonitoring the UV at 214 nm (2.0 A). A=0.1% TFA, B=0.1% TFA/50% ACN.Fractions 26-29 were combined frozen and lyophilized to give several mgof product. Analytical HPLC showed a purity of 90% and MALDI massspectral analysis confirmed a mass of 3515 for the desired product.

Example 10 Glucagon Cys²⁴ maleimido,PEG-3.4K-dimer

16 mg Glucagon Cys²⁴ and 1.02 mg Mal-PEG-Mal-3400,poly(ethyleneglycol)-bis-maleimide avg. M.W. 3400, (Nektar Therpeutics)were dissolved in 3.5 phosphate buffered saline and 0.5 ml 0.01M EDTAand the reaction was stirred at room temperature. After 16 hrs, another16 mg of Glucagon Cys²⁴ was added and the stirring continued. Afterapproximately 40 hrs, the reaction mixture was loaded onto a PharmciaPepRPC 16/10 column and an acetonitrile gradient was run on a PharmaciaFPLC while collecting 2 min fractions and monitoring the IJV at 214 nm(2.0 A). Flow=2 ml/min, A=0.1% TFA, B=0.1% TFA/50% ACN. Fractions 69-74were combined frozen and lyophilized to give 10.4 mg. Analytical HPLCshowed a purity of 90% and MALDI mass spectral analysis shows acomponent in the 9500-11,000 range which is consistent with the desireddimer.

Example 11 Synthesis of Glucagon Lactams

285 mg (0.2 mmole) methoxybenzhydrylamine resin (Midwest Biotech) wasadded to a 60 mL reaction vessels and the following sequence wasassembled on a modified Applied Biosystems 430A peptide synthesizerusing Boc DEPBT-activated single couplings.

(12-16 Lactam; SEQ ID NO: 12) HSQGTFTSDYSKYLDERRAQDFVQWLMNT-NH2

The following side chain protecting groups were used: Arg(Tos),Asp(OcHx),

Asn(Xan), Glu(OFm), His(BOM), Lys(Fmoc), Ser(Bzl), Thr(Bzl), Trp(CHO),Tyr(Br—Z). Lys(Cl—Z) was used at position 12 if lactams were constructedfrom 16-20, 20-24, or 24-28. The completed peptidyl resin was treatedwith 20% piperidine/dimethylformamide for one hour with rotation toremove the Trp formyl group as well as the Fmoc and OFm protection fromLys12 and Glu16. Upon confirmation of removal by a positive ninhydrintest, the resin was washed with dimethylformamide, followed bydichloromethane and than again with dimethylformamide. The resin wastreated with 520 mg (1 mmole)Benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate(PyBOP) in dimethylformamide and diisopropylethylamine (DIEA). Thereaction proceeded for 8-10 hours and the cyclization was confirmed by anegative ninhydrin reaction. The resin was washed withdimethylformamide, followed by dichloromethane and subsequently treatedwith trifluoroacetic acid for 10 minutes. The removal of the Boc groupwas confirmed by a positive ninhydrin reaction. The resin was washedwith dimethylformamide and dichloromethane and dried before beingtransferred to a hydrofluoric acid (HF) reaction vessel. 500 μL p-cresolwas added along with a magnetic stir bar. The vessel was attached to theHF apparatus (Peninsula Labs), cooled in a dry ice/methanol bath,evacuated, and approximately 10 mL of liquid hydrofluoric acid wascondensed into the vessel. The reaction was stirred for 1 hour in an icebath and the HF was subsequently removed in vacuo. The residue wassuspended in ethyl ether; the solids were filtered, washed with ether,and the peptide was solubilized with 150 mL 20% acetonitrile/1% aceticacid.

An analytical HPLC analysis of the crude solubilized peptide wasconducted under the following conditions [4.6×30 mm Xterra C8, 1.50mL/min, 220 nm, A buffer 0.1% TFA/10% ACN, B buffer 0.1% TFA/100% ACN,gradient 5-95% B over 15 minutes]. The extract was diluted twofold withwater and loaded onto a 2.2×25 cm Vydac C4 preparative reverse phasecolumn and eluted using an acetonitrile gradient on a Waters HPLC system(A buffer of 0.1% TFA/10% ACN, B buffer of 0.1% TFA/10% CAN and agradient of 0-100% B over 120 minutes at a flow of 15.00 ml/min. HPLCanalysis of the purified peptide demonstrated greater than 95% purityand electrospray ionization mass spectral analysis confirmed a mass of3506 Da for the 12-16 lactam. Lactams from 16-20, 20-24, and 24-28 wereprepared similarly.

Example 12 Glucagon Solubility Assays

A solution (1 mg/ml or 3 mg/ml) of glucagon (or an analog) is preparedin 0.01N HCl. 100 ul of stock solution is diluted to 1 ml with 0.01N HCland the UV absorbance (276 nm) is determined. The pH of the remainingstock solution is adjusted to 047 using 200-250 ul 0.1M Na₂HPO₄ (pH9.2).The solution is allowed to stand overnight at 4° C. then centrifuged.100 ul of supernatant is then diluted to 1 ml with 0.01N HCl, and the UVabsorbance is determined (in duplicate).

The initial absorbance reading is compensated for the increase in volumeand the following calculation is used to establish percent solubility:

${\frac{{Final}\mspace{14mu} {Absorbance}}{{Initial}\mspace{14mu} {Absorbance}} \times 100} = {{percent}\mspace{14mu} {soluble}}$

Results are shown in Table 1 wherein Glucagon-Cex represents wild typeglucagon (SEQ ID NO: 1) plus a carboxy terminal addition of SEQ ID NO:26 and Glucagon-Cex R¹² represents SEQ ID NO: 39.

TABLE 1 Solubility date for glucagon analogs Analog Percent SolubleGlucagon 16 Glucagon-Cex, R12 104 Glucagon-Cex 87 Oxyntomodulin 104Glucagon, Cys17PEG5K 94 Glucagon, Cys21PEG5K 105 Glucagon, Cys24PEG5K133

Example 13 Glucagon Receptor Binding Assay

The affinity of peptides to the glucagon receptor was measured in acompetition binding assay utilizing scintillation proximity assaytechnology. Serial 3-fold dilutions of the peptides made inscintillation proximity assay buffer (0.05 M Tris-HCl, pH 7.5, 0.15 MNaCl, 0.1% w/v bovine serum albumin) were mixed in 96 well white/clearbottom plate (Corning Inc., Acton, Mass.) with 0.05 nM(3-[¹²⁵I]-iodotyrosyl) Tyr10 glucagon (Amersham Biosciences, Piscataway,N.J.), 1-6 micrograms per well, plasma membrane fragments prepared fromcells over-expressing human glucagon receptor, and 1 mg/wellpolyethyleneimine-treated wheat germ agglutinin type A scintillationproximity assay beads (Amersham Biosciences, Piscataway, N.J.). Upon 5min shaking at 800 rpm on a rotary shaker, the plate was incubated 12 hat room temperature and then read on MicroBeta1450 liquid scintillationcounter (Perkin-Elmer, Wellesley, Mass.). Non-specifically bound (NSB)radioactivity was measured in the wells with 4 times greaterconcentration of “cold” native ligand than the highest concentration intest samples and total bound radioactivity was detected in the wellswith no competitor. Percent specific binding was calculated asfollowing: % Specific Binding=((Bound−NSB)/(Total bound−NSB))×100. IC₅₀values were determined by using Origin software (OriginLab, Northampton,Mass.).

Example 14 Functional Assay-cAMP Synthesis

The ability of glucagon analogs to induce cAMP was measured in a fireflyluciferase-based reporter assay. HEK293 cells co-transfected with eitherglucagon- or GLP-1 receptor and luciferase gene linked to cAMPresponsive element were serum deprived by culturing 16 h in DMEM(Invitrogen, Carlsbad, Calif.) supplemented with 0.25% Bovine GrowthSerum (HyClone, Logan, Utah) and then incubated with serial dilutions ofeither glucagon, GLP-1 or novel glucagon analogs for 5 h at 37° C., 5%CO₂ in 96 well poly-D-Lysine-coated “Biocoat” plates (BD Biosciences,San Jose, Calif.). At the end of the incubation 100 microliters ofLucLite luminescence substrate reagent (Perkin-Elmer, Wellesley, Mass.)were added to each well. The plate was shaken briefly, incubated 10 minin the dark and light output was measured on MicroBeta-1450 liquidscintillation counter (Perkin-Elmer, Wellesley, Mass.). Effective 50%concentrations were calculated by using Origin software (OriginLab,Northampton, Mass. Results are shown in FIGS. 3-9 and in Tables 2through 10.

TABLE 2 cAMP Induction by Glucagon Analogs with C-Terminus ExtensioncAMP Induction Glucagon Receptor GLP-1 Receptor Peptide EC₅₀, nM N*EC₅₀, nM N Glucagon 0.22 ± 0.09 14 3.85 ± 1.64 10 GLP-1 2214.00 ±182.43  2 0.04 ± 0.01 14 Glucagon Cex 0.25 ± 0.15 6 2.75 ± 2.03 7Oxyntomodulin 3.25 ± 1.65 5 2.53 ± 1.74 5 Oxyntomodulin KRNR 2.77 ± 1.744 3.21 ± 0.49 2 Glucagon R12 0.41 ± 0.17 6 0.48 ± 0.11 5 Glucagon R12Cex 0.35 ± 0.23 10 1.25 ± 0.63 10 Glucagon R12 K20 0.84 ± 0.40 5 0.82 ±0.49 5 Glucagon R12 K24 1.00 ± 0.39 4 1.25 ± 0.97 5 Glucagon R12 K290.81 ± 0.49 5 0.41 ± 0.24 6 Glucagon Amide 0.26 ± 0.15 3 1.90 ± 0.35 2Oxyntomodulin C24 2.54 ± 0.63 2 5.27 ± 0.26 2 Oxyntomodulin C24 PEG 0.97± 0.04 1 1.29 ± 0.11 1 20K *number of experiments

TABLE 3 cAMP Induction by Pegylated Glucagon Analogs cAMP InductionGlucagon Receptor GLP-1 Receptor Peptide EC₅₀, nM N* EC₅₀, nM N Glucagon0.33 ± 0.23 18 12.71 ± 3.74 2 Glucagon C17 PEG 5K 0.82 ± 0.15 4 55.86 ±1.13 2 Glucagon C21 PEG 5K 0.37 ± 0.16 6 11.52 ± 3.68 2 Glucagon C24 PEG5K 0.22 ± 0.10 12 13.65 ± 2.95 4 Glucagon C29 PEG 5K 0.96 ± 0.07 2 12.71± 3.74 2 Glucagon C24 PEG 20K 0.08 ± 0.05 3 Not determined Glucagon C24Dimer 0.10 ± 0.05 3 Not determined GLP-1 >1000  0.05 ± 0.02 4 *number ofexperiments

TABLE 4 cAMP Induction by E16 Glucagon Analogs Percent Potency Relativeto Native Ligand Peptide GRec GLP-1Rec E16 Gluc-NH2 187.2 17.8 Glucagon100.0 0.8 Gluc-NH2 43.2 4.0 NLeu3, E16 Gluc-NH2 7.6 20.6 E3, E16Gluc-NH2 1.6 28.8 Orn3, E16 Gluc-NH2 0.5 0.1 GLP-1 <0.1 100

TABLE 5 cAMP Induction by E16 Glucagon Analogs Percent Potency Relativeto Native Ligand Peptide GRec GLP-1Rec E16 Gluc-NH2 187.2 17.8 E15, E16Gluc-NH2 147.0 9.2 E16, K20 Gluc-NH2 130.1 41.5 Gluc-NH2 43.2 4.0

TABLE 6 EC50 values for cAMP Induction by E16 Glucagon Analogs GlucagonGLP-1 Receptor Receptor EC50 EC50 Peptide (nM) StDev n (nM) StDev nGlucagon 0.28 0.14 10 4.51 N/A 1 Glucagon-NH2 0.53 0.33 8 1.82 0.96 5E16 Gluc-NH2 0.07 0.07 10 0.16 0.14 10 E16, G30 Gluc-NH2 0.41 0.36 50.24 0.10 5 E16, G30 Gluc-Cex 0.51 0.46 5 1.19 0.86 5 GLP-1 2214 N/A 10.03 0.02 9

TABLE 7 EC50 values for cAMP Induction by E16 Glucagon Analogs GlucagonGLP-1 Receptor Receptor EC50 EC50 Peptide (nM) StDev n (nM) StDev n E16Glucagon NH2 0.07 0.07 10 0.16 0.14 10 hCSO₃16 Glucagon-NH2 0.25 0.12 20.19 0.02 2 hE16 Glucagon-NH2 0.17 0.08 2 0.25 0.03 2 H16 Glucagon-NH20.45 0.3 2 0.38 0.11 2 Q16 Glucagon-NH2 0.22 0.1 2 0.39 0.08 2 D16Glucagon-NH2 0.56 0.15 2 0.93 0.28 2 (S16) Glucagon-NH2 0.53 0.33 8 1.820.96 5

TABLE 8 EC50 values for cAMP Induction by E16 Glucagon Analogs GlucagonGLP-1 Receptor Receptor EC50 EC50 Peptide (nM) StD n (nM) StDev n E16Glucagon NH2 0.07 0.07 10 0.16 0.14 10 T16 Glucagon NH2 0.10 0.02 3 1.990.48 3 G16 Glucagon NH2 0.10 0.01 3 2.46 0.60 3 Glucagon NH2 0.53 0.33 41.82 0.96 5 GLP-1 2214 N/A 1 0.03 0.02 9 E16 Gluc NH₂ was 4-fold morepotent at the glucagon receptor relative to G16-COOH and T16 Gluc NH₂,when the compounds were tested side by side.

TABLE 9 cAMP Induction by E16/Lactam Glucagon Analogs Percent PotencyRelative to Native Ligand Peptide GRec GLP-1Rec E24K28 Gluc-NH2 Lac196.4 12.5 E16K20 Gluc-NH2 Lac 180.8 63.0 E12E16 Gluc-NH2 Lac 154.2 63.3K20E24 Gluc-NH2 Lac 120.2 8.1 E16 Gluc-NH2 187.2 17.8 E16, K20 Gluc-NH2130.1 41.5 Glucagon 100.0 0.8 Gluc-NH2 43.2 4.0

TABLE 10 cAMP Induction by GLP-1 17-26 Glucagon Analogs Glucagon GLP-1Receptor Receptor Peptide EC50(nM) StD EC50(nM) StD GLP-1 0.023 0.002Gluc-NH2 0.159 0.023 E16 GLP-1 0.009 0.000 E16 Glucagon-NH2 0.072 0.007E16 GLP(17-26)Glu(27-29)-NH2 0.076 0.004 0.014 0.001 E16 GLP(17-29)-NH20.46 0.023 0.010 0.000 E16 GLP(17-29)-NH2 E24, K28 0.23 0.020 0.007 E16GLP(17-29)-NH2 E24, K28 0.16 0.017 0.007 0.000 Lactam

Example 15 Stability Assay for Glucagon Cys-Maleimido PEG Analogs

Each glucagon analog was dissolved in water or PBS and an initial HPLCanalysis was conducted. After adjusting the pH (4, 5, 6, 7), the sampleswere incubated over a specified time period at 37° C. and re-analyzed byHPLC to determine the integrity of the peptide. The concentration of thespecific peptide of interest was determined and the percent remainingintact was calculated relative to the initial analysis. Results forGlucagon Cys²¹-maleimidoPEG_(5K) are shown in FIGS. 1 and 2.

Example 16

The following glucagon peptides are constructed generally as describedabove in Examples 1-11:

In all of the following sequences, “a” means a C-terminal amide.

(SEQ ID NO: 70) HSQGT FTSDY SKYLD ERRAQ DFVQW LMNTa (SEQ ID NO: 71)HSQGT FTSDY SKYLD ERRAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 72)HSQGT FTSDY SKYLD ERRAK DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 73)HSQGT FTSDY SKYLD ERRAQ DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 74)HSQGT FTSDY SKYLD ERRAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 75)HSQGT FTSDY SKYLD KRRAE DFVQW LMNTa (SEQ ID NO: 76)HSQGT FTSDY SKYLD ERAAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 77)HSQGT FTSDY SKYLD ERAAK DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 78)HSQGT FTSDY SKYLD ERAAQ DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 79)HSQGT FTSDY SKYLD ERAAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 80)HSQGT FTSDY SKYLD KRAAE DFVQW LMNTa (SEQ ID NO: 81)HSQGT FTSDY SKYLD EQAAK EFIAW LMNTa (lactam @ 12-16; SEQ ID NO: 82)HSQGT FTSDY SKYLD EQAAK EFIAW LMNTa (lactam @ 16-20; SEQ ID NO: 83)HSQGT FTSDY SKYLD EQAAK EFIAW LMNTa (SEQ ID NO: 84)HSQGT FTSDY SKYLD EQAAK EFIAW LVKGa (lactam @ 12-16; SEQ ID NO: 85)HSQGT FTSDY SKYLD EQAAK EFIAW LVKGa (lactam @ 16-20; SEQ ID NO: 86)HSQGT FTSDY SKYLD EQAAK EFIAW LVKGa (SEQ ID NO: 87)X1SQGT FTSDY SKYLD ERRAQ DFVQW LMNTa (SEQ ID NO: 88)X1SQGT FTSDY SKYLD ERRAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 89)X1SQGT FTSDY SKYLD ERRAK DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 90)X1SQGT FTSDY SKYLD ERRAQ DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 91)X1SQGT FTSDY SKYLD ERRAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 92)X1SQGT FTSDY SKYLD KRRAE DFVQW LMNTa (SEQ ID NO: 93)X1SQGT FTSDY SKYLD ERAAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 94)X1SQGT FTSDY SKYLD ERAAK DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 95)X1SQGT FTSDY SKYLD ERAAQ DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 96)X1SQGT FTSDY SKYLD ERAAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 97)X1SQGT FTSDY SKYLD KRAAE DFVQW LMNTa (SEQ ID NO: 98)X1SQGT FTSDY SKYLD EQAAK EFIAW LMNTa (lactam @ 12-16; SEQ ID NO: 99)X1SQGT FTSDY SKYLD EQAAK EFIAW LMNTa (lactam @ 16-20; SEQ ID NO: 100)X1SQGT FTSDY SKYLD EQAAK EFTAW LMNTa (SEQ ID NO: 101)X1SQGT FTSDY SKYLD EQAAK EFIAW LVKGa (lactam @ 12-16; SEQ ID NO: 102)X1SQGT FTSDY SKYLD EQAAK EFIAW LVKGa (lactam @ 16-20; SEQ ID NO: 103)X1SQGT FTSDY SKYLD EQAAK EFIAW LVKGaWherein in the preceding sequences, X1=(Des-amino)His

(SEQ ID NO: 104) HX2QGT FTSDY SKYLD ERRAQ DFVQW LMNTa (SEQ ID NO: 105)HX2QGT FTSDY SKYLD ERRAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 106)HX2QGT FTSDY SKYLD ERRAK DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 107)HX2QGT FTSDY SKYLD ERRAQ DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 108)HX2QGT FTSDY SKYLD ERRAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 109)HX2QGT FTSDY SKYLD KRRAE DFVQW LMNTa (SEQ ID NO: 110)HX2QGT FTSDY SKYLD ERAAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 111)HX2QGT FTSDY SKYLD ERAAK DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 112)HX2QGT FTSDY SKYLD ERAAQ DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 113)HX2QGT FTSDY SKYLD ERAAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 114)HX2QGT FTSDY SKYLD KRAAE DFVQW LMNTa (SEQ ID NO: 115)HX2QGT FTSDY SKYLD EQAAK EFIAW LMNTa (lactam @ 12-16; SEQ ID NO: 116)HX2QGT FTSDY SKYLD EQAAK EFIAW LMNTa (lactam @ 16-20; SEQ ID NO: 117)HX2QGT FTSDY SKYLD EQAAK EFIAW LMNTa (SEQ ID NO: 118)HX2QGT FTSDY SKYLD EQAAK EFIAW LVKGa (lactam @ 12-16; SEQ ID NO: 119)HX2QGT FTSDY SKYLD EQAAK EFIAW LVKGa (lactam @ 16-20; SEQ ID NO: 120)HX2QGT FTSDY SKYLD EQAAK EFIAW LVKGa Wherein in the preceding sequences X2=Aminoisobutyric acid

(SEQ ID NO: 121) HX2QGT FTSDY SKYLD ERRAQ DFVQW LMNTa (SEQ ID NO: 122)HX2QGT FTSDY SKYLD ERRAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 123)HX2QGT FTSDY SKYLD ERRAK DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 124)HX2QGT FTSDY SKYLD ERRAQ DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 125)HX2QGT FTSDY SKYLD ERRAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 126)HX2QGT FTSDY SKYLD KRRAE DFVQW LMNTa (SEQ ID NO: 127)HX2QGT FTSDY SKYLD ERAAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 128)HX2QGT FTSDY SKYLD ERAAK DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 129)HX2QGT FTSDY SKYLD ERAAQ DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 130)HX2QGT FTSDY SKYLD ERAAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 131)HX2QGT FTSDY SKYLD KRAAE DFVQW LMNTa (SEQ ID NO: 132)HX2QGT FTSDY SKYLD EQAAK EFIAW LMNTa (lactam @ 12-16; SEQ ID NO: 133)HX2QGT FTSDY SKYLD EQAAK EFIAW LMNTa (lactam @ 16-20; SEQ ID NO: 134)HX2QGT FTSDY SKYLD EQAAK EFIAW LMNTa (SEQ ID NO: 135)HX2QGT FTSDY SKYLD EQAAK EFIAW LVKGa (lactam @ 12-16; SEQ ID NO: 136)HX2QGT FTSDY SKYLD EQAAK EFIAW LVKGa (lactam @ 16-20; SEQ ID NO: 137)HX2QGT FTSDY SKYLD EQAAK EFIAW LVKGaWherein in the preceding sequences X2=(D-Ala)

(SEQ ID NO: 138) HSEGT FTSDY SKYLD ERRAQ DFVQW LMNTa (SEQ ID NO: 139)HSEGT FTSDY SKYLD ERRAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 140)HSEGT FTSDY SKYLD ERRAK DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 141)HSEGT FTSDY SKYLD ERRAQ DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 142)HSEGT FTSDY SKYLD ERRAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 143)HSEGT FTSDY SKYLD KRRAE DFVQW LMNTa (SEQ ID NO: 144)HSEGT FTSDY SKYLD ERAAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 145)HSEGT FTSDY SKYLD ERAAK DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 146)HSEGT FTSDY SKYLD ERAAQ DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 147)HSEGT FTSDY SKYLD ERAAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 148)HSEGT FTSDY SKYLD KRAAE DFVQW LMNTa (SEQ ID NO: 149)HSEGT FTSDY SKYLD EQAAK EFIAW LMNTa (lactam @ 12-16; SEQ ID NO: 150)HSEGT FTSDY SKYLD EQAAK EFIAW LMNTa (lactam @ 16-20; SEQ ID NO: 151)HSEGT FTSDY SKYLD EQAAK EFIAW LMNTa (SEQ ID NO: 152)HSEGT FTSDY SKYLD EQAAK EFIAW LVKGa (lactam @ 12-16; SEQ ID NO: 153)HSEGT FTSDY SKYLD EQAAK EFIAW LVKGa (lactam @ 16-20; SEQ ID NO: 154)HSEGT FTSDY SKYLD EQAAK EFIAW LVKGa (SEQ ID NO: 155)X1SEGT FTSDY SKYLD ERRAQ DFVQW LMNTa (SEQ ID NO: 156)X1SEGT FTSDY SKYLD ERRAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 157)X1SEGT FTSDY SKYLD ERRAK DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 158)X1SEGT FTSDY SKYLD ERRAQ DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 159)X1SEGT FTSDY SKYLD ERRAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 160)X1SEGT FTSDY SKYLD KRRAE DFVQW LMNTa (SEQ ID NO: 161)X1SEGT FTSDY SKYLD ERAAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 162)X1SEGT FTSDY SKYLD ERAAK DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 163)X1SEGT FTSDY SKYLD ERAAQ DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 164)X1SEGT FTSDY SKYLD ERAAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 165)X1SEGT FTSDY SKYLD KRAAE DFVQW LMNTa (SEQ ID NO: 166)X1SEGT FTSDY SKYLD EQAAK EFIAW LMNTa (lactam @ 12-16; SEQ ID NO: 167)X1SEGT FTSDY SKYLD EQAAK EFIAW LMNTa (lactam @ 16-20; SEQ ID NO: 168)X1SEGT FTSDY SKYLD EQAAK EFIAW LMNTa (SEQ ID NO: 169)X1SEGT FTSDY SKYLD EQAAK EFIAW LVKGa (lactam @ 12-16; SEQ ID NO: 170)X1SEGT FTSDY SKYLD EQAAK EFIAW LVKGa (lactam @ 16-20; SEQ ID NO: 171)X1SEGT FTSDY SKYLD EQAAK EFIAW LVKGaWherein in the preceding sequences X1=(Des-amino)His

(SEQ ID NO: 172) HX2EGT FTSDY SKYLD ERRAQ DFVQW LMNTa (SEQ ID NO: 173)HX2EGT FTSDY SKYLD ERRAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 174)HX2EGT FTSDY SKYLD ERRAK DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 175)HX2EGT FTSDY SKYLD ERRAQ DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 176)HX2EGT FTSDY SKYLD ERRAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 177)HX2EGT FTSDY SKYLD KRRAE DFVQW LMNTa (SEQ ID NO: 178)HX2EGT FTSDY SKYLD ERAAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 179)HX2EGT FTSDY SKYLD ERAAK DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 180)HX2EGT FTSDY SKYLD ERAAQ DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 181)HX2EGT FTSDY SKYLD ERAAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 182)HX2EGT FTSDY SKYLD KRAAE DFVQW LMNTa (SEQ ID NO: 183)HX2EGT FTSDY SKYLD EQAAK EFIAW LMNTa (lactam @ 12-16; SEQ ID NO: 184)HX2EGT FTSDY SKYLD EQAAK EFIAW LMNTa (lactam @ 16-20; SEQ ID NO: 185)HX2EGT FTSDY SKYLD EQAAK EFIAW LMNTa (SEQ ID NO: 186)HX2EGT FTSDY SKYLD EQAAK EFIAW LVKGa (lactam @ 12-16; SEQ ID NO: 187)HX2EGT FTSDY SKYLD EQAAK EFIAW LVKGa (lactam @ 16-20; SEQ ID NO: 188)HX2EGT FTSDY SKYLD EQAAK EFIAW LVKGaWherein in the preceding sequences X2=Aminoisobutyric acid

(SEQ ID NO: 189) HX2EGT FTSDY SKYLD ERRAQ DFVQW LMNTa (SEQ ID NO: 190)HX2EGT FTSDY SKYLD ERRAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 191)HX2EGT FTSDY SKYLD ERRAK DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 192)HX2EGT FTSDY SKYLD ERRAQ DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 193)HX2EGT FTSDY SKYLD ERRAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 194)HX2EGT FTSDY SKYLD KRRAE DFVQW LMNTa (SEQ ID NO: 195)HX2EGT FTSDY SKYLD ERAAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 196)HX2EGT FTSDY SKYLD ERAAK DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 197)HX2EGT FTSDY SKYLD ERAAQ DFVQW LMNTa (lactam @ 12-16; SEQ ID NO: 198)HX2EGT FTSDY SKYLD ERAAK DFVQW LMNTa (lactam @ 16-20; SEQ ID NO: 199)HX2EGT FTSDY SKYLD KRAAE DFVQW LMNTa (SEQ ID NO: 200)HX2EGT FTSDY SKYLD EQAAK EFIAW LMNTa (lactam @ 12-16; SEQ ID NO: 201)HX2EGT FTSDY SKYLD EQAAK EFIAW LMNTa (lactam @ 16-20; SEQ ID NO: 202)HX2EGT FTSDY SKYLD EQAAK EFIAW LMNTa (SEQ ID NO: 203)HX2EGT FTSDY SKYLD EQAAK EFIAW LVKGa (lactam @ 12-16; SEQ ID NO: 204)HX2EGT FTSDY SKYLD EQAAK EFIAW LVKGa (lactam @ 16-20; SEQ ID NO: 205)HX2EGT FTSDY SKYLD EQAAK EFIAW LVKGaWherein in the preceding sequences X2=(D-Ala)

(SEQ ID NO: 206) HSQGT FTSDY SKYLD ERRAQ DFVC*W LMNTa (SEQ ID NO: 207)HSQGT FTSDY SKYLD ERRAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 208)HSQGT FTSDY SKYLD ERRAK DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 209)HSQGT FTSDY SKYLD ERRAQ DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 210)HSQGT FTSDY SKYLD ERRAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 211)HSQGT FTSDY SKYLD KRRAE DFVC*W LMNTa (SEQ ID NO: 212)HSQGT FTSDY SKYLD ERAAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 213)HSQGT FTSDY SKYLD ERAAK DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 214)HSQGT FTSDY SKYLD ERAAQ DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 215)HSQGT FTSDY SKYLD ERAAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 216)HSQGT FTSDY SKYLD KRAAE DFVC*W LMNTa (SEQ ID NO: 217)HSQGT FTSDY SKYLD EQAAK EFIC*W LMNTa (lactam @ 12-16; SEQ ID NO: 218)HSQGT FTSDY SKYLD EQAAK EFIC*W LMNTa (lactam @ 16-20; SEQ ID NO: 219)HSQGT FTSDY SKYLD EQAAK EFIC*W LMNTa (SEQ ID NO: 220)HSQGT FTSDY SKYLD EQAAK EFIC*W LVKGa (lactam @ 12-16; SEQ ID NO: 221)HSQGT FTSDY SKYLD EQAAK EFIC*W LVKGa (lactam @ 16-20; SEQ ID NO: 222)HSQGT FTSDY SKYLD EQAAK EFIC*W LVKGa (SEQ ID NO: 223)X1SQGT FTSDY SKYLD ERRAQ DFVC*W LMNTa (SEQ ID NO: 224)X1SQGT FTSDY SKYLD ERRAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 225)X1SQGT FTSDY SKYLD ERRAK DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 226)X1SQGT FTSDY SKYLD ERRAQ DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 227)X1SQGT FTSDY SKYLD ERRAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 228)X1SQGT FTSDY SKYLD KRRAE DFVC*W LMNTa (SEQ ID NO: 229)X1SQGT FTSDY SKYLD ERAAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 230)X1SQGT FTSDY SKYLD ERAAK DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 231)X1SQGT FTSDY SKYLD ERAAQ DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 232)X1SQGT FTSDY SKYLD ERAAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 233)X1SQGT FTSDY SKYLD KRAAE DFVC*W LMNTa (SEQ ID NO: 234)X1SQGT FTSDY SKYLD EQAAK EFIC*W LMNTa (lactam @ 12-16; SEQ ID NO: 235)X1SQGT FTSDY SKYLD EQAAK EFIC*W LMNTa (lactam @ 16-20; SEQ ID NO: 236)X1SQGT FTSDY SKYLD EQAAK EFIC*W LMNTa (SEQ ID NO: 237)X1SQGT FTSDY SKYLD EQAAK EFIC*W LVKGa (lactam @ 12-16; SEQ ID NO: 238)X1SQGT FTSDY SKYLD EQAAK EFIC*W LVKGa (lactam @ 16-20; SEQ ID NO: 239)X1SQGT FTSDY SKYLD EQAAK EFIC*W LVKGaWherein in the preceding sequences X1=(Des-amino)His; and wherein the C*is a Cys, or a Cys attached to a hydrophilic polymer, or alternativelythe C* is a Cys attached to a polyethylene glycol of about 20 kD averageweight, or alternatively the C* is a Cys attached to a polyethyleneglycol of about 40 kD average weight.

(SEQ ID NO: 240) HX2QGT FTSDY SKYLD ERRAQ DFVC*W LMNTa (SEQ ID NO: 241)HX2QGT FTSDY SKYLD ERRAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 242)HX2QGT FTSDY SKYLD ERRAK DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 243)HX2QGT FTSDY SKYLD ERRAQ DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 244)HX2QGT FTSDY SKYLD ERRAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 245)HX2QGT FTSDY SKYLD KRRAE DFVC*W LMNTa (SEQ ID NO: 246)HX2QGT FTSDY SKYLD ERAAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 247)HX2QGT FTSDY SKYLD ERAAK DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 248)HX2QGT FTSDY SKYLD ERAAQ DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 249)HX2QGT FTSDY SKYLD ERAAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 250)HX2QGT FTSDY SKYLD KRAAE DFVC*W LMNTa (SEQ ID NO: 251)HX2QGT FTSDY SKYLD EQAAK EFIC*W LMNTa (lactam @ 12-16; SEQ ID NO: 252)HX2QGT FTSDY SKYLD EQAAK EFIC*W LMNTa (lactam @ 16-20; SEQ ID NO: 253)HX2QGT FTSDY SKYLD EQAAK EFIC*W LMNTa (SEQ ID NO: 254)HX2QGT FTSDY SKYLD EQAAK EFIC*W LVKGa (lactam @ 12-16; SEQ ID NO: 255)HX2QGT FTSDY SKYLD EQAAK EFIC*W LVKGa (lactam @ 16-20; SEQ ID NO: 256)HX2QGT FTSDY SKYLD EQAAK EFIC*W LVKGaWherein in the preceding sequences X2=Aminoisobutyric acid; and whereinthe C* is a Cys, or a Cys attached to a hydrophilic polymer, oralternatively the C* is a Cys attached to a polyethylene glycol of about20 kD average weight, or alternatively the C* is a Cys attached to apolyethylene glycol of about 40 kD average weight.

(SEQ ID NO: 257) HX2QGT FTSDY SKYLD ERRAQ DFVC*W LMNTa (SEQ ID NO: 258)HX2QGT FTSDY SKYLD ERRAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 259)HX2QGT FTSDY SKYLD ERRAK DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 260)HX2QGT FTSDY SKYLD ERRAQ DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 261)HX2QGT FTSDY SKYLD ERRAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 262)HX2QGT FTSDY SKYLD KRRAE DFVC*W LMNTa (SEQ ID NO: 263)HX2QGT FTSDY SKYLD ERAAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 264)HX2QGT FTSDY SKYLD ERAAK DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 265)HX2QGT FTSDY SKYLD ERAAQ DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 266)HX2QGT FTSDY SKYLD ERAAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 267)HX2QGT FTSDY SKYLD KRAAE DFVC*W LMNTa (SEQ ID NO: 268)HX2QGT FTSDY SKYLD EQAAK EFIC*W LMNTa (lactam @ 12-16; SEQ ID NO: 269)HX2QGT FTSDY SKYLD EQAAK EFIC*W LMNTa (lactam @ 16-20; SEQ ID NO: 270)HX2QGT FTSDY SKYLD EQAAK EFIC*W LMNTa (SEQ ID NO: 271)HX2QGT FTSDY SKYLD EQAAK EFIC*W LVKGa (lactam @ 12-16; SEQ ID NO: 272)HX2QGT FTSDY SKYLD EQAAK EFIC*W LVKGa (lactam @ 16-20; SEQ ID NO: 273)HX2QGT FTSDY SKYLD EQAAK EFIC*W LVKGaWherein in the preceding sequences X2=(D-Ala); and wherein the C* is aCys, or a Cys attached to a hydrophilic polymer, or alternatively the C*is a Cys attached to a polyethylene glycol of about 20 kD averageweight, or alternatively the C* is a Cys attached to a polyethyleneglycol of about 40 kD average weight.

(SEQ ID NO: 274) HSEGT FTSDY SKYLD ERRAQ DFVC*W LMNTa (SEQ ID NO: 275)HSEGT FTSDY SKYLD ERRAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 276)HSEGT FTSDY SKYLD ERRAK DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 277)HSEGT FTSDY SKYLD ERRAQ DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 278)HSEGT FTSDY SKYLD ERRAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 279)HSEGT FTSDY SKYLD KRRAE DFVC*W LMNTa (SEQ ID NO: 280)HSEGT FTSDY SKYLD ERAAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 281)HSEGT FTSDY SKYLD ERAAK DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 282)HSEGT FTSDY SKYLD ERAAQ DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 283)HSEGT FTSDY SKYLD ERAAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 284)HSEGT FTSDY SKYLD KRAAE DFVC*W LMNTa (SEQ ID NO: 285)HSEGT FTSDY SKYLD EQAAK EFIC*W LMNTa (lactam @ 12-16; SEQ ID NO: 286)HSEGT FTSDY SKYLD EQAAK EFIC*W LMNTa (lactam @ 16-20; SEQ ID NO: 287)HSEGT FTSDY SKYLD EQAAK EFIC*W LMNTa (SEQ ID NO: 288)HSEGT FTSDY SKYLD EQAAK EFIC*W LVKGa (lactam @ 12-16; SEQ ID NO: 289)HSEGT FTSDY SKYLD EQAAK EFIC*W LVKGa (lactam @ 16-20; SEQ ID NO: 290)HSEGT FTSDY SKYLD EQAAK EFIC*W LVKGa (SEQ ID NO: 291)X1SEGT FTSDY SKYLD ERRAQ DFVC*W LMNTa (SEQ ID NO: 292)X1SEGT FTSDY SKYLD ERRAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 293)X1SEGT FTSDY SKYLD ERRAK DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 294)X1SEGT FTSDY SKYLD ERRAQ DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 295)X1SEGT FTSDY SKYLD ERRAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 296)X1SEGT FTSDY SKYLD KRRAE DFVC*W LMNTa (SEQ ID NO: 297)X1SEGT FTSDY SKYLD ERAAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 298)X1SEGT FTSDY SKYLD ERAAK DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 299)X1SEGT FTSDY SKYLD ERAAQ DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 300)X1SEGT FTSDY SKYLD ERAAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 301)X1SEGT FTSDY SKYLD KRAAE DFVC*W LMNTa (SEQ ID NO: 302)X1SEGT FTSDY SKYLD EQAAK EFIC*W LMNTa (lactam @ 12-16; SEQ ID NO: 303)X1SEGT FTSDY SKYLD EQAAK EFIC*W LMNTa (lactam @ 16-20; SEQ ID NO: 304)X1SEGT FTSDY SKYLD EQAAK EFIC*W LMNTa (SEQ ID NO: 305)X1SEGT FTSDY SKYLD EQAAK EFIC*W LVKGa (lactam @ 12-16; SEQ ID NO: 306)X1SEGT FTSDY SKYLD EQAAK EFIC*W LVKGa (lactam @ 16-20; SEQ ID NO: 307)X1SEGT FTSDY SKYLD EQAAK EFIC*W LVKGaWherein in the preceding sequences X1=(Des-amino)His; and wherein the C*is a Cys, or a Cys attached to a hydrophilic polymer, or alternativelythe C* is a Cys attached to a polyethylene glycol of about 20 kD averageweight, or alternatively the C* is a Cys attached to a polyethyleneglycol of about 40 kD average weight.

(SEQ ID NO: 308) HX2EGT FTSDY SKYLD ERRAQ DFVC*W LMNTa (SEQ ID NO: 309)HX2EGT FTSDY SKYLD ERRAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 310)HX2EGT FTSDY SKYLD ERRAK DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 311)HX2EGT FTSDY SKYLD ERRAQ DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 312)HX2EGT FTSDY SKYLD ERRAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 313)HX2EGT FTSDY SKYLD KRRAE DFVC*W LMNTa (SEQ ID NO: 314)HX2EGT FTSDY SKYLD ERAAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 315)HX2EGT FTSDY SKYLD ERAAK DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 316)HX2EGT FTSDY SKYLD ERAAQ DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 317)HX2EGT FTSDY SKYLD ERAAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 318)HX2EGT FTSDY SKYLD KRAAE DFVC*W LMNTa (SEQ ID NO: 319)HX2EGT FTSDY SKYLD EQAAK EFIC*W LMNTa (lactam @ 12-16; SEQ ID NO: 320)HX2EGT FTSDY SKYLD EQAAK EFIC*W LMNTa (lactam @ 16-20; SEQ ID NO: 321)HX2EGT FTSDY SKYLD EQAAK EFIC*W LMNTa (SEQ ID NO: 322)HX2EGT FTSDY SKYLD EQAAK EFIC*W LVKGa (lactam @ 12-16; SEQ ID NO: 323)HX2EGT FTSDY SKYLD EQAAK EFIC*W LVKGa (lactam @ 16-20; SEQ ID NO: 324)HX2EGT FTSDY SKYLD EQAAK EFIC*W LVKGaWherein in the preceding sequences X2=Aminoisobutyric acid; and whereinthe C* is a Cys, or a Cys attached to a hydrophilic polymer, oralternatively the C* is a Cys attached to a polyethylene glycol of about20 Id) average weight, or alternatively the C* is a Cys attached to apolyethylene glycol of about 40 kD average weight.

(SEQ ID NO: 325) HX2EGT FTSDY SKYLD ERRAQ DFVC*W LMNTa (SEQ ID NO: 326)HX2EGT FTSDY SKYLD ERRAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 327)HX2EGT FTSDY SKYLD ERRAK DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 328)HX2EGT FTSDY SKYLD ERRAQ DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 329)HX2EGT FTSDY SKYLD ERRAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 330)HX2EGT FTSDY SKYLD KRRAE DFVC*W LMNTa (SEQ ID NO: 331)HX2EGT FTSDY SKYLD ERAAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 332)HX2EGT FTSDY SKYLD ERAAK DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 333)HX2EGT FTSDY SKYLD ERAAQ DFVC*W LMNTa (lactam @ 12-16; SEQ ID NO: 334)HX2EGT FTSDY SKYLD ERAAK DFVC*W LMNTa (lactam @ 16-20; SEQ ID NO: 335)HX2EGT FTSDY SKYLD KRAAE DFVC*W LMNTa (SEQ ID NO: 336)HX2EGT FTSDY SKYLD EQAAK EFIC*W LMNTa (lactam @ 12-16; SEQ ID NO: 337)HX2EGT FTSDY SKYLD EQAAK EFIC*W LMNTa (lactam @ 16-20; SEQ ID NO: 338)HX2EGT FTSDY SKYLD EQAAK EFIC*W LMNTa (SEQ ID NO: 339)HX2EGT FTSDY SKYLD EQAAK EFIC*W LVKGa (lactam @ 12-16; SEQ ID NO: 340)HX2EGT FTSDY SKYLD EQAAK EFIC*W LVKGa  (lactam @ 16-20; SEQ ID NO: 341)HX2EGT FTSDY SKYLD EQAAK EFIC*W LVKGa Wherein in the preceding sequences X2=(D-Ala); and wherein the C* is aCys, or a Cys attached to a hydrophilic polymer, or alternatively the C*is a Cys attached to a polyethylene glycol of about 20 kD averageweight, or alternatively the C* is a Cys attached to a polyethyleneglycol of about 40 kD average weight.

(SEQ ID NO: 342) HSQGT FTSDY SKYLD C*RRAK DFVQW LMNTa (SEQ ID NO: 343)HSQGT FTSDY SKYLD C*RAAK DFVQW LMNTa (SEQ ID NO: 344)HSQGT FTSDY SKYLD C*QAAK EFIAW LMNTa (SEQ ID NO: 345)HSQGT FTSDY SKYLD C*QAAK EFIAW LVKGa (SEQ ID NO: 346)X1SQGT FTSDY SKYLD C*RRAK DFVQW LMNTa (SEQ ID NO: 347)X1SQGT FTSDY SKYLD C*RAAK DFVQW LMNTa (SEQ ID NO: 348)X1SQGT FTSDY SKYLD C*QAAK EFIAW LMNTa (SEQ ID NO: 349)X1SQGT FTSDY SKYLD C*QAAK EFIAW LVKGaWherein X1=(Des-amino)His; and wherein the C* is a Cys, or a Cysattached to a hydrophilic polymer, or alternatively the C* is a Cysattached to a polyethylene glycol of about 20 kD average weight, oralternatively the C* is a Cys attached to a polyethylene glycol of about40 kD average weight.

(SEQ ID NO: 350) HX2QGT FTSDY SKYLD C*RRAK DFVQW LMNTa (SEQ ID NO: 351)HX2QGT FTSDY SKYLD C*RAAK DFVQW LMNTa (SEQ ID NO: 352)HX2QGT FTSDY SKYLD C*QAAK EFIAW LMNTa (SEQ ID NO: 353)HX2QGT FTSDY SKYLD C*QAAK EFIAW LVKGaWherein X2=Aminoisobutyric acid; and wherein the C* is a Cys, or a Cysattached to a hydrophilic polymer, or alternatively the C* is a Cysattached to a polyethylene glycol of about 20 kD average weight, oralternatively the C* is a Cys attached to a polyethylene glycol of about40 kD average weight.

(SEQ ID NO: 354) HX2QGT FTSDY SKYLD C*RRAK DFVQW LMNTa (SEQ ID NO: 355)HX2QGT FTSDY SKYLD C*RAAK DFVQW LMNTa (SEQ ID NO: 356)HX2QGT FTSDY SKYLD C*QAAK EFIAW LMNTa (SEQ ID NO: 357)HX2QGT FTSDY SKYLD C*QAAK EFIAW LVKGaWherein X2=(D-Ala); and wherein the C* is a Cys, or a Cys attached to ahydrophilic polymer, or alternatively the C* is a Cys attached to apolyethylene glycol of about 20 kD average weight, or alternatively theC* is a Cys attached to a polyethylene glycol of about 40 kD averageweight.

(SEQ ID NO: 358) HSEGT FTSDY SKYLD C*RRAK DFVQW LMNTa (SEQ ID NO: 359)HSEGT FTSDY SKYLD C*RAAK DFVQW LMNTa (SEQ ID NO: 360)HSEGT FTSDY SKYLD C*QAAK EFIAW LMNTa (SEQ ID NO: 361)HSEGT FTSDY SKYLD C*QAAK EFIAW LVKGa (SEQ ID NO: 362)X1SEGT FTSDY SKYLD C*RRAK DFVQW LMNTa (SEQ ID NO: 363)X1SEGT FTSDY SKYLD C*RAAK DFVQW LMNTa (SEQ ID NO: 364)X1SEGT FTSDY SKYLD C*QAAK EFIAW LMNTa (SEQ ID NO: 365)X1SEGT FTSDY SKYLD C*QAAK EFIAW LVKGaWherein X1=(Des-amino)His; and wherein the C* is a Cys, or a Cysattached to a hydrophilic polymer, or alternatively the C* is a Cysattached to a polyethylene glycol of about 20 kD average weight, oralternatively the C* is a Cys attached to a polyethylene glycol of about40 kD average weight.

(SEQ ID NO: 366) HX2EGT FTSDY SKYLD C*RRAK DFVQW LMNTa (SEQ ID NO: 367)HX2EGT FTSDY SKYLD C*RAAK DFVQW LMNTa (SEQ ID NO: 368)HX2EGT FTSDY SKYLD C*QAAK EFIAW LMNTa (SEQ ID NO: 369)HX2EGT FTSDY SKYLD C*QAAK EFIAW LVKGa Wherein X2=(D-Ala); and wherein the C* is a Cys, or a Cys attached to ahydrophilic polymer, or alternatively the C* is a Cys attached to apolyethylene glycol of about 20 kD average weight, or alternatively theC* is a Cys attached to a polyethylene glycol of about 40 kD averageweight.

(SEQ ID NO: 370) HX2EGT FTSDY SKYLD C*RRAK DFVQW LMNTa (SEQ ID NO: 371)HX2EGT FTSDY SKYLD C*RAAK DFVQW LMNTa (SEQ ID NO: 372)HX2EGT FTSDY SKYLD C*QAAK EFIAW LMNTa (SEQ ID NO: 373)HX2EGT FTSDY SKYLD C*QAAK EFIAW LVKGaWherein X2=(D-Ala); and wherein the C* is a Cys, or a Cys attached to ahydrophilic polymer, or alternatively the C* is a Cys attached to apolyethylene glycol of about 20 kD average weight, or alternatively theC* is a Cys attached to a polyethylene glycol of about 40 kD averageweight.

(SEQ ID NO: 374) HSQGT FTSDY SKYLD ERRAQ DFVQW LMDTa (SEQ ID NO: 375)HSQGT FTSDY SKYLD ERRAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 376)HSQGT FTSDY SKYLD ERRAK DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 377)HSQGT FTSDY SKYLD ERRAQ DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 378)HSQGT FTSDY SKYLD ERRAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 379)HSQGT FTSDY SKYLD KRRAE DFVQW LMDTa (SEQ ID NO: 380)HSQGT FTSDY SKYLD ERAAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 381)HSQGT FTSDY SKYLD ERAAK DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 382)HSQGT FTSDY SKYLD ERAAQ DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 383)HSQGT FTSDY SKYLD ERAAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 384)HSQGT FTSDY SKYLD KRAAE DFVQW LMDTa (SEQ ID NO: 385)HSQGT FTSDY SKYLD EQAAK EFIAW LMDTa (lactam @ 12-16; SEQ ID NO: 386)HSQGT FTSDY SKYLD EQAAK EFIAW LMDTa (lactam @ 16-20; SEQ ID NO: 387)HSQGT FTSDY SKYLD EQAAK EFIAW LMDTa (SEQ ID NO: 388)X1SQGT FTSDY SKYLD ERRAQ DFVQW LMDTa (SEQ ID NO: 389)X1SQGT FTSDY SKYLD ERRAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 390)X1SQGT FTSDY SKYLD ERRAK DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 391)X1SQGT FTSDY SKYLD ERRAQ DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 392)X1SQGT FTSDY SKYLD ERRAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 393)X1SQGT FTSDY SKYLD KRRAE DFVQW LMDTa (SEQ ID NO: 394)X1SQGT FTSDY SKYLD ERAAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 395)X1SQGT FTSDY SKYLD ERAAK DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 396)X1SQGT FTSDY SKYLD ERAAQ DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 397)X1SQGT FTSDY SKYLD ERAAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 398)X1SQGT FTSDY SKYLD KRAAE DFVQW LMDTa (SEQ ID NO: 399)X1SQGT FTSDY SKYLD EQAAK EFIAW LMDTa (lactam @ 12-16; SEQ ID NO: 400)X1SQGT FTSDY SKYLD EQAAK EFIAW LMDTa (lactam @ 16-20; SEQ ID NO: 401)X1SQGT FTSDY SKYLD EQAAK EFIAW LMDTaWherein in the preceding sequences X1=(Des-amino)His

(SEQ ID NO: 402) HX2QGT FTSDY SKYLD ERRAQ DFVQW LMDTa (SEQ ID NO: 403)HX2QGT FTSDY SKYLD ERRAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 404)HX2QGT FTSDY SKYLD ERRAK DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 405)HX2QGT FTSDY SKYLD ERRAQ DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 406)HX2QGT FTSDY SKYLD ERRAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 407)HX2QGT FTSDY SKYLD KRRAE DFVQW LMDTa (SEQ ID NO: 408)HX2QGT FTSDY SKYLD ERAAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 409)HX2QGT FTSDY SKYLD ERAAK DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 410)HX2QGT FTSDY SKYLD ERAAQ DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 411)HX2QGT FTSDY SKYLD ERAAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 412)HX2QGT FTSDY SKYLD KRAAE DFVQW LMDTa (SEQ ID NO: 413)HX2QGT FTSDY SKYLD EQAAK EFIAW LMDTa (lactam @ 12-16; SEQ ID NO: 414)HX2QGT FTSDY SKYLD EQAAK EFIAW LMDTa (lactam @ 16-20; SEQ ID NO: 415)HX2QGT FTSDY SKYLD EQAAK EFIAW LMDTaWherein in the preceding sequences X2=Aminoisobutyric acid

(SEQ ID NO: 416) HX2QGT FTSDY SKYLD ERRAQ DFVQW LMDTa (SEQ ID NO: 417)HX2QGT FTSDY SKYLD ERRAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 418)HX2QGT FTSDY SKYLD ERRAK DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 419)HX2QGT FTSDY SKYLD ERRAQ DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 420)HX2QGT FTSDY SKYLD ERRAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 421)HX2QGT FTSDY SKYLD KRRAE DFVQW LMDTa (SEQ ID NO: 422)HX2QGT FTSDY SKYLD ERAAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 423)HX2QGT FTSDY SKYLD ERAAK DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 424)HX2QGT FTSDY SKYLD ERAAQ DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 425)HX2QGT FTSDY SKYLD ERAAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 426)HX2QGT FTSDY SKYLD KRAAE DFVQW LMDTa (SEQ ID NO: 427)HX2QGT FTSDY SKYLD EQAAK EFIAW LMDTa (lactam @ 12-16; SEQ ID NO: 428)HX2QGT FTSDY SKYLD EQAAK EFIAW LMDTa (lactam @ 16-20; SEQ ID NO: 429)HX2QGT FTSDY SKYLD EQAAK EFIAW LMDTa Wherein in the preceding sequences X2=(D-Ala)

(SEQ ID NO: 430) HSEGT FTSDY SKYLD ERRAQ DFVQW LMDTa (SEQ ID NO: 431)HSEGT FTSDY SKYLD ERRAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 432)HSEGT FTSDY SKYLD ERRAK DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 433)HSEGT FTSDY SKYLD ERRAQ DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 434)HSEGT FTSDY SKYLD ERRAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 435)HSEGT FTSDY SKYLD KRRAE DFVQW LMDTa (SEQ ID NO: 436)HSEGT FTSDY SKYLD ERAAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 437)HSEGT FTSDY SKYLD ERAAK DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 438)HSEGT FTSDY SKYLD ERAAQ DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 439)HSEGT FTSDY SKYLD ERAAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 440)HSEGT FTSDY SKYLD KRAAE DFVQW LMDTa (SEQ ID NO: 441)HSEGT FTSDY SKYLD EQAAK EFIAW LMDTa (lactam @ 12-16; SEQ ID NO: 442)HSEGT FTSDY SKYLD EQAAK EFIAW LMDTa (lactam @ 16-20; SEQ ID NO: 443)HSEGT FTSDY SKYLD EQAAK EFIAW LMDTa (SEQ ID NO: 444)X1SEGT FTSDY SKYLD ERRAQ DFVQW LMDTa (SEQ ID NO: 445)X1SEGT FTSDY SKYLD ERRAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 446)X1SEGT FTSDY SKYLD ERRAK DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 447)X1SEGT FTSDY SKYLD ERRAQ DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 448)X1SEGT FTSDY SKYLD ERRAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 449)X1SEGT FTSDY SKYLD KRRAE DFVQW LMDTa (SEQ ID NO: 450)X1SEGT FTSDY SKYLD ERAAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 451)X1SEGT FTSDY SKYLD ERAAK DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 452)X1SEGT FTSDY SKYLD ERAAQ DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 453)X1SEGT FTSDY SKYLD ERAAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 454)X1SEGT FTSDY SKYLD KRAAE DFVQW LMDTa (SEQ ID NO: 455)X1SEGT FTSDY SKYLD EQAAK EFIAW LMDTa (lactam @ 12-16; SEQ ID NO: 456)X1SEGT FTSDY SKYLD EQAAK EFIAW LMDTa (lactam @ 16-20; SEQ ID NO: 457)X1SEGT FTSDY SKYLD EQAAK EFIAW LMDTaWherein in the preceding sequences X1=(Des-amino)His

(SEQ ID NO: 458) HX2EGT FTSDY SKYLD ERRAQ DFVQW LMDTa (SEQ ID NO: 459)HX2EGT FTSDY SKYLD ERRAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 460)HX2EGT FTSDY SKYLD ERRAK DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 461)HX2EGT FTSDY SKYLD ERRAQ DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 462)HX2EGT FTSDY SKYLD ERRAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 463)HX2EGT FTSDY SKYLD KRRAE DFVQW LMDTa (SEQ ID NO: 464)HX2EGT FTSDY SKYLD ERAAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 465)HX2EGT FTSDY SKYLD ERAAK DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 466)HX2EGT FTSDY SKYLD ERAAQ DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 467)HX2EGT FTSDY SKYLD ERAAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 468)HX2EGT FTSDY SKYLD KRAAE DFVQW LMDTa (SEQ ID NO: 469)HX2EGT FTSDY SKYLD EQAAK EFIAW LMDTa (lactam @ 12-16; SEQ ID NO: 470)HX2EGT FTSDY SKYLD EQAAK EFIAW LMDTa (lactam @ 16-20; SEQ ID NO: 471)HX2EGT FTSDY SKYLD EQAAK EFIAW LMDTaWherein in the preceding sequences X2=Aminoisobutyric acid

(SEQ ID NO: 472) HX2EGT FTSDY SKYLD ERRAQ DFVQW LMDTa (SEQ ID NO: 473)HX2EGT FTSDY SKYLD ERRAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 474)HX2EGT FTSDY SKYLD ERRAK DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 475)HX2EGT FTSDY SKYLD ERRAQ DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 476)HX2EGT FTSDY SKYLD ERRAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 477)HX2EGT FTSDY SKYLD KRRAE DFVQW LMDTa (SEQ ID NO: 478)HX2EGT FTSDY SKYLD ERAAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 479)HX2EGT FTSDY SKYLD ERAAK DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 480)HX2EGT FTSDY SKYLD ERAAQ DFVQW LMDTa (lactam @ 12-16; SEQ ID NO: 481)HX2EGT FTSDY SKYLD ERAAK DFVQW LMDTa (lactam @ 16-20; SEQ ID NO: 482)HX2EGT FTSDY SKYLD KRAAE DFVQW LMDTa (SEQ ID NO: 483)HX2EGT FTSDY SKYLD EQAAK EFIAW LMDTa (lactam @ 12-16; SEQ ID NO: 484)HX2EGT FTSDY SKYLD EQAAK EFIAW LMDTa (lactam @ 16-20; SEQ ID NO: 485)HX2EGT FTSDY SKYLD EQAAK EFIAW LMDTaWherein in the preceding sequences X2=(D-Ala)The following glucagon peptides with a GLP-1/glucagon activity ratio ofabout 5 or more are also constructed generally as described above inExamples 1-11. Generally, in these peptides, AIB at position 2 providesDPP IV resistance but also significantly reduces glucagon activity.

(SEQ ID NO: 486) HX2QGT FTSDY SKYLD EQAAK EFIC*W LMNTa (SEQ ID NO: 487)HX2QGT FTSDY SKYLD EQAAK EFIAW LMNC*a (SEQ ID NO: 488)HX2QGT FTSDY SKYLD EQAAK EFIAW LMNGG PSSGA PPPSC*a (lactam @16-20; SEQ ID NO: 489) HX2QGT FTSDY SKYLD EQAAK EFIAW LMNGG PSSGAPPPSC*a (SEQ ID NO: 490)HX2QGT FTSDY SKYLD EQAAK EFIC*W LMNGG PSSGA PPPSa (lactam @16-20; SEQ ID NO: 491) HX2QGT FTSDY SKYLD EQAAK EFIC*W LMNGG PSSGA PPPSaWherein in the preceding sequences X2=AIB, and wherein the C* is a Cys,or a Cys attached to a hydrophilic polymer, or alternatively the C* is aCys attached to a polyethylene glycol of about 20 kD average weight, oralternatively the C* is a Cys attached to a polyethylene glycol of about40 kD average weight.

(SEQ ID NO: 492) HX2QGT FTSDY SKYLD ERAAK DFVC*W LMNTa (SEQ ID NO: 493)HX2QGT FTSDY SKYLD ERAAK DFVQW LMNC*a (SEQ ID NO: 494)HX2QGT FTSDY SKYLD ERAAK DFVQW LMNGG PSSGA PPPSC*a (lactam @16-20; SEQ ID NO: 495) HX2QGT FTSDY SKYLD ERAAK DFVQW LMNGG PSSGAPPPSC*a (SEQ ID NO: 496)HX2QGT FTSDY SKYLD ERAAK DFVC*W LMNGG PSSGA PPPSa (lactam @16-20; SEQ ID NO: 497) HX2QGT FTSDY SKYLD ERAAK DFVC*W LMNGG PSSGA PPPSa(SEQ ID NO: 498) HX2QGT FTSDY SKYLD ERRAK DFVC*W LMNTa (SEQ ID NO: 499)HX2QGT FTSDY SKYLD ERRAK DFVQW LMNC*a (SEQ ID NO: 500)HX2QGT FTSDY SKYLD ERRAK DFVQW LMNGG PSSGA PPPSC*a (lactam @16-20; SEQ ID NO: 501) HX2QGT FTSDY SKYLD ERRAK DFVQW LMNGG PSSGAPPPSC*a (SEQ ID NO: 502)HX2QGT FTSDY SKYLD ERRAK DFVC*W LMNGG PSSGA PPPSa (lactam @16-20; SEQ ID NO: 503) HX2QGT FTSDY SKYLD ERRAK DFVC*W LMNGG PSSGA PPPSaWherein in the preceding sequences X2=AIB, and wherein the C* is a Cys,or a Cys attached to a hydrophilic polymer, or alternatively the C* is aCys attached to a polyethylene glycol of about 20 kD average weight, oralternatively the C* is a Cys attached to a polyethylene glycol of about40 kD average weight.The following glucagon peptides which are GLP-1/glucagon co-agonists arealso constructed generally as described above in Examples 1-11.Formation of a lactam bridge between amino acids 16 and 20 restores thereduction in glucagon activity caused by the substitution at position 2.

(lactam @ 16-20; SEQ ID NO: 504) HX2QGT FTSDY SKYLD EQAAK EFIC*W LMNTaWherein in the preceding sequence X2=AIB, and wherein the C* is a Cys,or a Cys attached to a hydrophilic polymer, or alternatively the C* is aCys attached to a polyethylene glycol of about 20 kD average weight, oralternatively the C* is a Cys attached to a polyethylene glycol of about40 kD average weight.

(lactam @ 16-20; SEQ ID NO: 505) X1SQGT FTSDY SKYLD EQAAK EFIC*W LMNTa(lactam @ 16-20; SEQ ID NO: 506) X1SQGT FTSDY SKYLD EQAAK EFIAW LMNC*a(lactam @ 16-20; SEQ ID NO: 507)X1SQGT FTSDY SKYLD EQAAK EFIAW LMNGG PSSGA PPPSC*a (lactam @16-20; SEQ ID NO: 508) X1SQGT FTSDY SKYLD ERRAK DFVQW LMNGG PSSGAPPPSC*a (lactam @ 16-20; SEQ ID NO: 509)X1SQGT FTSDY SKYLD EQAAK EFIC*W LMNGG PSSGA PPPSa (lactam @16-20; SEQ ID NO: 510) X1SQGT FTSDY SKYLD ERRAK DEVC*W LMNTa (lactam @16-20; SEQ ID NO: 511) HX2QGT FTSDY SKYLD ERRAK DFVC*W LMNTa (lactam @16-20; SEQ ID NO: 512) X1SQGT FTSDY SKYLD ERRAK DFVQW LMNC*a (lactam @16-20; SEQ ID NO: 513) X1SQGT FTSDY SKYLD ERRAK DFVC*W LMNGG PSSGA PPPSaWherein in the preceding sequences X1=DMEA (alpha, alpha-dimethylimidiazole acetic acid), and wherein the C* is a Cys, or a Cys attachedto a hydrophilic polymer, or alternatively the C* is a Cys attached to apolyethylene glycol of about 20 kD average weight, or alternatively theC* is a Cys attached to a polyethylene glycol of about 40 kD averageweight.

(optionally with lactam @ 16-20; SEQ  ID NO: 514)HSQGT FTSDY SKYLD EQAAK EFIC*W LMNTaWherein the C* is a Cys, or a Cys attached to a hydrophilic polymer, oralternatively the C* is a Cys attached to a polyethylene glycol of about20 kD average weight, or alternatively the C* is a Cys attached to apolyethylene glycol of about 40 kD average weight.

(lactam @ 16-20; SEQ ID NO: 517) HX2QGT FTSDY SKYLD ERRAK DFVC*W LMNTa(lactam @ 16-20; SEQ ID NO: 528) HX2QGT FTSDY SKYLD ERRAK DFVC*W LMNTa(SEQ ID NO: 531) HX2QGT FTSDY SKYLD ERRAK EFIC*W LMNGG PSSGA PPPSC*a(SEQ ID NO: 532) HX2QGT FTSDY SKYLD EQAAK EFIAW LMNGG PSSGA PPPSC*C*a(SEQ ID NO: 533) HX2QGT FTSDY SKYLD EQAAK EFIC*W LMNGG PSSGA PPPSaWherein in the preceding sequence X2=AIB, and wherein the C* is a Cys,or a Cys attached to a hydrophilic polymer, or alternatively the C* is aCys attached to a polyethylene glycol of about 20 kD average weight, oralternatively the C* is a Cys attached to a polyethylene glycol of about40 kD average weight.

(SEQ ID NO: 518) HSQGT FTSDYSKYLD EQAAK EFIC*W LMNTa (SEQ ID NO: 519)X1SQGT FTSDYSKYLD EQAAK EFIC*W LMNTa (SEQ ID NO: 520)X1SQGT FTSDYSKYLD EQAAK EFIAW LMNC*a (SEQ ID NO: 529)X1SQGT FTSDY SKYLD ERRAK DFVC*W LMNGG PSSGA PPPSa (SEQ ID NO: 530)X1SQGT FTSDY SKYLD ERRAK DFVC*W LMNTa

Wherein in the preceding sequences X1=DMIA (alpha, alpha-dimethylimidiazole acetic acid), and wherein the C* is a Cys, or a Cys attachedto a hydrophilic polymer, or alternatively the C* is a Cys attached to apolyethylene glycol of about 20 kD average weight, or alternatively theC* is a Cys attached to a polyethylene glycol of about 40 kD averageweight.

(SEQ ID NO: 521) HSQGT FTSDYSKYLD SRRAQ DFVQW LMNTGPSSGAPPPSa(SEQ ID NO: 522) HSQGT FTSDYSKYLD SRRAQ DFVQW LMNGGPSSGAPPPSa(SEQ ID NO: 523) HSQGT FTSDYSKYLD SRRAQ DFVQW LMKGGPSSGAPPPSa(SEQ ID NO: 524) HSQGT FTSDYSKYLD SRRAQ DFVQW LVKGGPSSGAPPPSa(SEQ ID NO: 525) HSQGT FTSDYSKYLD SRRAQ DFVQW LMDGGPSSGAPPPSa(SEQ ID NO: 526) HSQGT FTSDYSKYLD ERRAK DFVQW LMDGGPSSGAPPPSa(SEQ ID NO: 527) HAEGT FTSDV SSYLE GQAAK EFIAW LVKGGa (SEQ ID NO: 61)X1X2QGT FTSDY SKYLD ERX5AK DFVX3W LMNX4whereinX1=His, D-histidine, desaminohistidine, hydroxyl-histidine,acetyl-histidine, homo-histidine or alpha, alpha-dimethyl imidiazoleacetic acid (DMIA) N-methyl histidine, alpha-methyl histidine, orimidazole acetic acid,X2=Ser, D-serine, Ala, Val, glycine, N-methyl serine or aminoisobutyricacid (AIB), N-methyl alanine and D-alanine.

X3=Ala, Gln or Cys-PEG X4=Thr-CONH₂ or Cys-PEG or GGPSSGAPPPS (SEQ IDNO: 515) or GGPSSGAPPPSC-PEG (SEQ ID NO: 516)

Provided that when X3 is Cys-PEG, X4 is not Cys-PEG or GGPSSGAPPPSC-PEG(SEQ ID NO: 516), and when X2=Ser, X1 is not His.

X5=Ala or Arg

(SEQ ID NO: 62) X1X2QGT FTSDY SKYLD EQ X5AK EFI X3W LMNX4whereinX1=His, D-histidine, desaminohistidine, hydroxyl-histidine,acetyl-histidine, homo-histidine or alpha, alpha-dimethyl imidiazoleacetic acid (DHEA), N-methyl histidine, alpha-methyl histidine, orimidazole acetic acidX2=Ser, D-serine, Ala, Val, glycine, N-methyl serine or aminoisobutyricacid (AIB), N-methyl alanine and D-alanine.

X3=Ala, Gln or Cys-PEG X4=Thr-CONH2 or Cys-PEG or GGPSSGAPPPS (SEQ IDNO: 515) or GGPSSGAPPPSC-PEG (SEQ ID NO: 516)

Provided that when X3 is Cys-PEG, X4 is not Cys-PEG or GGPSSGAPPPSC-PEG(SEQ ID NO: 516), and when X2=Ser, X1 is not His.

X5=Ala or Arg

Any of the preceding sequences can include additional modifications,e.g., 1, 2, 3, 4 or 5 modifications that do not destroy activity,including but not limited to W10 or R20 substitutions that can be usedto enhance potency. Any of the preceding sequences can also be producedwithout the modifications that confer DPP IV resistance, i.e. in whichthe native His is at position 1 and the native Ser is at position 2. Inaddition, any of the preceding compounds may optionally be linked to aconjugate, such as a heterologous polypeptide, an immunoglobulin or aportion thereof (e.g. Fc region), a targeting agent, a diagnostic label,or a diagnostic or therapeutic agent.

Example 17

The following glucagon peptides modified to comprise the c-terminalextension of SEQ ID NO: 26 linked to the carboxy terminus of theglucagon peptide were constructed generally as described above inExamples 1-11 and assayed for activity at the GLP-1 and glucagonreceptors using the in vitro assay described in Example 14.

Table 11 represents the activity of various glucagon analogs at theglucagon and GLP-1 receptors. The data shows that for glucagon analogscomprising the c-terminal extension of SEQ ID NO: 26, amino acidsubstititions at positions 16, 20, 28 and 29 can impact the analogsactivity at the GLP-1 receptor.

TABLE 11 Glucagon-Cex Structure Activity Relationship Glucagon ReceptorGLP 1 Receptor EC50 Relative Relative Glucagon Peptide (nM) Potency (%)EC50 (nM) Potency (%) -MNT²⁹ (SEQ ID NO: 1)  0.086 100 -MNTG³⁰ PSSGAPPPS0.14 61 1.19 2 (SEQ ID NO: 521) -MNGG³⁰ PSSGAPPPS 0.28 30 0.31 8(SEQ ID NO: 522) -MKGG³⁰ PSSGAPPPS 0.61 14 0.80 3 (SEQ ID NO: 523)-VKGG³⁰ PSSGAPPPS 1.16 7 0.21 12 (SEQ ID NO: 524) -MDGG³⁰ PSSGAPPPS 0.1272 0.13 19 (SEQ ID NO: 525) E¹⁶K²⁰-MDGG³⁰ PSSGAPPPS 0.22 39 0.020 125(SEQ ID NO: 526) GLP-1-VKGG³⁰ 0.025 100 (SEQ ID NO: 527)

Example 18

Table 12 represents in vitro data accumulated for various glucagonpeptides comparing their relative activities at the glucagon and GLP-1receptors.

TABLE 12 COMPARISON OF AGONISTS AND CO-AGONISTS w/ and w/o PEG % PotencyRelative to Native CONTROLS GR GL-1R Glucagon 100 0.78 GLP-1 <0.01 100Parent w/o PEG Parent w/PEG % Potency % Potency Relative to Relative toNative Native GR GLP-1R GR GLP-1R AGONISTS Chimera AIB2, Cys24 (SEQ IDNO: 486) 15.4 160.6 2.6 82.5 Chimera AIB2, Cys29 (SEQ ID NO: 487) 20.1124.6 5.6 54.3 Chimera AIB2, Gly29,30 Cys40 Cex (SEQ ID NO: 2.2 359.10.3 68.8 488) Chimera AIB2, Gly29,30 Cys40 Cex Lactam (SEQ ID 14.2 169.63.2 63.6 NO: 489) Chimera AIB2, Gly29,30 Cys24 Cex (SEQ ID NO: 2.5 457.80.2 95.4 490) Chimera AIB2, Gly29,30 Cys24 Cex Lactam (SEQ ID 25.2 381.51.4 96.4 NO: 491) E16, K20AIB2, A18 Cys24 (SEQ ID NO: 492) — — 1.1 73.5E16, K20AIB2, A18 Gly29,30 Cys24 Cex (SEQ ID — — 0.1 88.5 NO: 496)CO-AGONISTS Chimera DMIA1, Cys24 Lactam (SEQ ID NO: 505) 160.7 82.5 19.112.5 Chimera AIB2, Cys24 Lactam (SEQ ID NO: 504) 114.2 230.4 9.2 38.0Chimera DMIA1, Cys29 Lactam (SEQ ID NO: 506) — — — — Chimera DMIA1,Gly29,30 Cys40 Cex Lactam (SEQ — — — — ID NO: 507) E16, K20 DMIA1Gly29,30 Cys40 Cex — — — — Lactam (SEQ ID NO: 508) Chimera DMIA1,Gly29,30 Cys24 Cex Lactam (SEQ — — — — ID NO: 509) E16, K20 DMIA1, Cys24Lactam (SEQ ID NO: 510) — — 64.1 9.3 E16, K20 AIB2, Cys24 Lactam (SEQ IDNO: 517) 108.3 96.9 15.8 31.0 Chimera Cys24 (SEQ ID NO: 518) — — 19.829.3 E16, K20 DMIA1, Gly29,30 Cys24 Cex 116.0 78.3 12.6 11.3 Lactam (SEQID NO: 513) Chimera DMIA1, Cys29 (SEQ ID NO: 520) — — 5.3 27.3 ChimeraDMIA1, Cys24 (SEQ ID NO: 519) 28.9 64.5 6.9 19.3

1.-65. (canceled)
 66. A glucagon peptide comprising the amino acid sequence of: (SEQ ID NO: 61) X1X2QGT FTSDY SKYLD ERX5AK DFVX3W LMNX4 or (SEQ ID NO: 62) X1X2QGT FTSDY SKYLD EQ X5AK EFI X3W LMNX4

wherein X1=His, D-histidine, desaminohistidine, hydroxyl-histidine, acetyl-histidine, homo-histidine or alpha, alpha-dimethyl imidiazole acetic acid (DMIA) N-methyl histidine, alpha-methyl histidine, or imidazole acetic acid, X2=Ser, D-serine, Ala, Val, glycine, N-methyl serine or aminoisobutyric acid (AIB), N-methyl alanine and D-alanine. X3=Ala, Gln or Cys-PEG X4=Thr-CONH2 or Cys-PEG or GGPSSGAPPPS (SEQ ID NO: 515) or GGPSSGAPPPSC-PEG (SEQ ID NO: 516) X5=Ala or Arg wherein, when X3 is Cys-PEG, X4 is not Cys-PEG or GGPSSGAPPPSC-PEG (SEQ ID NO: 516), and when X2=Ser, X1 is not His.
 67. The glucagon peptide of claim 66, wherein X₃ is Cys-PEG.
 68. The glucagon peptide of claim 67, wherein X₄ is Thr-CONH2.
 69. The glucagon peptide of claim 66, wherein X1 is His and X2 is AIB or X1 is DMIA and X2 is Ser.
 70. The glucagon peptide of claim 66, which further comprises a lactam bridge between amino acids at positions 16 and
 20. 71. A glucagon peptide comprising the sequence of SEQ ID NO: 55, wherein: the amino acid at position 1 or 2 is modified to exhibit reduced susceptibility to cleavage by dipeptidyl peptidase IV, wherein the amino acid at position 1 is selected from the group consisting of d-histidine, desaminohistidine, hydroxyl-histidine, acetyl-histidine homo-histidine, N-methyl histidine, alpha-methyl histidine, imidazole acetic acid, and alpha, alpha-dimethyl imidiazole acetic acid (DMIA) or the amino acid at position 2 is selected from the group consisting of d-serine, alanine, D-alanine, valine, glycine, N-methyl serine, N-methyl alanine, and amino isobutyric acid; the amino acid at position 3 is Glu or Gln; the amino acid at position 10 is Tyr; the amino acid at position 12 is Lys; the amino acid at position 15 is Asp; the amino acid at position 16 is glutamic acid, the amino acid at position 20 is lysine, the amino acid at position 24 is Cys; the amino acid at position 27 is Met; the amino acid at position 28 is Asn; the amino acid at position 29 is Thr; the C-terminal carboxylic acid group is replaced with an amide, and a hydrophilic moiety is covalently bound at position 17, 21 or 24 or at the C-terminal amino acid of the glucagon peptide; wherein said glucagon peptide exhibits enhanced activity at the GLP-1 receptor, relative to native glucagon.
 72. The glucagon peptide of claim 71, comprising a lactam bridge between the glutamic acid at position 16 and the lysine at position
 20. 73. The glucagon peptide of claim 71, wherein said hydrophilic moiety is a polyethylene glycol chain.
 74. An analog of the glucagon amino acid sequence of SEQ ID NO: 1 that comprises an intramolecular bridge between the side chains of the amino acids at positions 12 and 16, 16 and 20, 20 and 24, or 24 and 28, wherein the carboxylic acid of the C-terminal amino acid is replaced with a charge-neutral group, wherein the amino acid at position 3 is glutamine or glutamic acid; wherein the amino acid at position 1 or 2 is modified to exhibit susceptibility to cleavage by DPP-IV; comprising a hydrophilic moiety covalently bound at position 17, 21, or 24, or at the C-terminal amino acid of the glucagon peptide; wherein the analog exhibits enhanced activity at the GLP-1 receptor as compared to native glucagon.
 75. (canceled)
 76. The analog of claim 74, comprising an intramolecular bridge between the side chains of the amino acids at positions 12 and 16 or 16 and
 20. 77. (canceled)
 78. The analog of claim 74, wherein the charge neutral group is an amide.
 79. A pharmaceutical composition comprising the glucagon peptide of claim 66, or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
 80. A pharmaceutical composition comprising the glucagon peptide of claim 71, or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
 81. A pharmaceutical composition comprising the glucagon peptide of claim 74, or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
 82. A method of treating diabetes or reducing weight in a subject in need thereof, said method comprising administering an effective amount of a pharmaceutical composition of claim
 79. 83. A method of treating diabetes or reducing weight in a subject in need thereof, said method comprising administering an effective amount of a pharmaceutical composition of claim
 80. 84. A method of treating diabetes or reducing weight in a subject in need thereof, said method comprising administering an effective amount of a pharmaceutical composition of claim
 81. 85. A glucagon peptide comprising the amino acid sequence of: (SEQ ID NO: 61) X1X2QGT FTSDY SKYLD ERX5AK DFVX3W LMNX4

wherein X1=alpha, alpha-dimethyl imidiazole acetic acid (DMIA), X2=Ser, X3=Cys-PEG X4=Thr-CONH2 X5=Arg wherein the glucagon peptide comprises a lactam bridge between the amino acids at positions 16 and
 20. 86. A pharmaceutical composition comprising the glucagon peptide of claim 85, or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
 87. A method of treating diabetes or reducing weight in a subject in need thereof, said method comprising administering an effective amount of a pharmaceutical composition of claim
 86. 